JP2004520262A - HIV-1 vaccine and screening method thereof - Google Patents

Abstract

Heterologous to HIV-1 in animals, preferably humans, using a modified HIV-1 envelope protein or fragment or a DNA encoding a modified HIV-1 envelope protein or fragment, wherein the HIV-1 envelope protein V2 region is deleted. An immunization method for raising an immune response, and an immunogenic pharmaceutical composition are provided. A humoral response to the heterologous HIV-1 strain is obtained.

Description

【００４８】
中和活性を１：１０の希釈液で評価し、ＤＮＡベクターのみでワクチン接種した動物から集めた血清で記録された非特異的中和を考慮に入れた（詳しくは材料及び方法を参照されたい）。（−）： ５０％の特異的中和が記録されなかった。（＋）： ５０％〜８０％の特異的中和が記録された。結果は３回の独立した中和実験からのものである。
Ａ１の動物を除いて、他の動物はすべて９２ＵＳ７１４に対して中和抗体を産生し、Ａ２とＡ５の動物のみが９２ＨＴ５９３に対して中和抗体を産生した。対照的に、修飾ΔＶ２ｇｐ１２０免疫原で免疫した６匹の動物の中から４匹が試験したほとんどの異種分離物に対して交差反応性中和抗体を産生した。更に、修飾免疫原で免疫した動物から集めた血清の中和の強さは、非修飾免疫原で免疫した動物から集めた血清より大きかった（上記表１を参照されたい）。従って、感染の８０％阻止は前者の血清でしばしば記録されたが、このレベルの阻止は２つの場合でのみ記録された（９２ＵＳ７１４と９２ＨＴ５９３の分離物に対してＡ５の動物からの血清）。
【００４９】
修飾ΔＶ２ｇｐ１４０免疫原でワクチン接種したアカゲザルにおける抗体の産生： 上記結果は、ワクチン惹起抗体の防御の可能性を結局は評価し得る動物モデルのアカゲザルにおける非修飾ＳＦ１６２ｇｐ１４０抗原と修飾ΔＶ２ｇｐ１４０抗原の免疫原の可能性の評価に駆り立てた。サルにこれらの２つの免疫原をＤＮＡの初回刺激に続いてタンパク質の追加ワクチン接種方法を用いてワクチン接種した。
エンベロープ特異抗体は、２回目のＤＮＡ免疫後に検出可能であった（図８）。この段階で、修飾抗原で免疫した動物における終点ＥＬＩＳＡ力価（Ｊ４０８とＨ４４５の動物）は１：２，０００の程度であった。対照的に、非修飾エンベロープで免疫した動物（Ｎ４７２とＰ６５５の動物）においては、抗体は動物Ｎ４７２でのみ検出可能であった（１：５００の程度での終点ＥＬＩＳＡ力価）。Ｈ４４５の動物を除いて、３回目のＤＮＡ免疫化によって抗体力価は高められなかった。抗ｇｐ１２０抗体と抗ｇｐ４１抗体はＤＮＡ免疫化で同調して産生した。
引き続き５〜１０ヶ月の所見では、非修飾ＳＦ１６２ｇｐ１４０免疫原で免疫した動物において抗体は検出不可能であり、修飾ΔＶ２ｇｐ１４０免疫原で免疫した動物においては、抗体は常に検出可能であるが、その力価は経時低下した。
【００５０】
ＤＮＡ＋タンパク質‘ブースター’免疫後、抗体力価はすべての動物において著しく増大した。ピーク値で（‘追加刺激’後の２〜４週間以内に達する）、修飾ΔＶ２ｇｐ１４０免疫原で免疫した動物における終点ＥＬＩＳＡ抗体力価はＪ４０８の動物については１：３０，０００、Ｈ４４５の動物については１：１１０，０００であった。力価は経時徐々に低下し、両動物共に数週間約１：８，０００で安定であった。非修飾ＳＦ１６２ｇｐ免疫原でワクチン接種した動物においては高いピーク抗体力価が記録された（終点ＥＬＩＳＡ抗体力価がＮ４７２の動物では１：１５０，０００、Ｐ６５５の動物では１７５，０００）。次の７週間の所見では、両動物共に抗体力価が約１：３５，０００まで急速に低下した。従って、ウサギで記録されたものと対照的に、サルにおいては非修飾免疫原は修飾免疫原より高い力価の結合抗体を産生した。
予想されるように、抗ＨＩＶエンベロープ抗体はＤＮＡベクターのみで免疫した対照動物（Ｍ８４４とＨ４７３）には産生されなかった。
同種ＳＦ１６２ΔＶ２分離物と親ＳＦ１６２分離物に対するサル血清の中和活性： ＤＮＡ相の免疫化では、修飾ΔＶ２ｇｐ１４０で免疫した動物のみがＳＦ１６２ウイルスとＳＦ１６２ΔＶ２ウイルスに対して中和抗体を惹起した（図９Ａ−Ｂ）。２回目のＤＮＡ免疫後、Ｊ４０８の動物は同種ＳＦ１６２ΔＶ２分離物に対して中和抗体を産生したが、親ＳＦ１６２分離物に対しては産生しなかった（図９Ａ）。Ｊ４０８の動物における中和抗体の力価は、３回目のＤＮＡ免疫後増大し、その点で両分離物の中和が記録されたが、結合抗体の力価は同時に増大しなかった（図９Ｂ）。対照的に、Ｈ４４５の動物においてＳＦ１６２ΔＶ２ウイルスに対しては非常に弱い中和抗体応答が惹起され、ＳＦ１６２ウイルスに対しては中和抗体応答が惹起されなかったが、この動物はＪ４０８で産生したのと同様の力価の結合抗体を産生した（図９Ｂ）。
【００５１】
ＤＮＡ＋タンパク質‘ブースター’免疫の２週間後、いずれの免疫原で免疫した動物から集めた血清もＳＦ１６２ΔＶ２感染を阻止した。修飾免疫原で免疫した動物から集めた血清の中和の強さは、非修飾免疫原で免疫した動物から集めた血清より大きかった。例えば、ＳＦ１６２ΔＶ２感染の５０％阻害は前者の血清から１：２，０００〜１：５，０００の希釈液で記録されたが、このレベルの阻害は後者の血清から集めた血清においてはこの希釈液で記録されなかった。いずれのΔＶ２ｇｐ１４０免疫動物も親ＳＦ１６２ウイルスに対して強い中和抗体を産生したが、ＳＦ１６２ｇｐ１４０免疫原で免疫した２匹の動物のうち１匹（Ｎ４７２）だけがこのウイルスに対して中和抗体を産生した。次の２週間にこれらの血清の中和の強さの変化は記録されなかったが、この期間中に抗体力価レベルの変化は検出可能であった（図９）。ベクターのみでワクチン接種した対照動物（Ｍ８４４とＨ４７３）は、中和抗体を産生しなかった。
サル血清による異種ＨＩＶ−１一次分離物の中和： 修飾免疫原と非修飾免疫原で免疫したサルに惹起された中和抗体応答の幅は、異種クレードＢ ＨＩＶ−１一次分離物の感染を阻止するこれらの２つの免疫原で免疫したサルから集めた血清の能力を比較することにより評価した。これらの分離物が種々のＭＡｂによって中和される感受性は、以前に報告されている（Ｄ’Ｓｏｕｚａ， Ｍ． Ｐ．， Ｄ． Ｌｉｖｎａｔ， Ｊ． Ａ． Ｂｒａｄａｃ ＆ Ｓ． Ｈ． Ｂｒｉｄｇｅｓ． １９９７． Ｅｖａｌｕａｔｉｏｎ ｏｆ ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｉｅｓ ｔｏ ｈｕｍａｎ ｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙ ｖｉｒｕｓ ｔｙｐｅ １ ｐｒｉｍａｒｙ ｉｓｏｌａｔｅｓ ｂｙ ｎｅｕｔｒａｌｉｚａｔｉｏｎ ａｓｓａｙｓ： ｐｅｒｆｏｒｍａｎｃｅ ｃｒｉｔｅｒｉａ ｆｏｒ ｓｅｌｅｃｔｉｎｇ ｃａｎｄｉｄａｔｅ ａｎｔｉｂｏｄｉｅｓ ｆｏｒ ｃｌｉｎｉｃａｌ ｔｒｉａｌｓ． ＡＩＤＳ Ｃｌｉｎｉｃａｌ Ｔｒｉａｌｓ Ｇｒｏｕｐ Ａｎｔｉｂｏｄｙ Ｓｅｌｅｃｔｉｏｎ Ｗｏｒｋｉｎｇ Ｇｒｏｕｐ． Ｊ． Ｉｎｆｅｃｔ． Ｄｉｓ． １７５：１０５６−６２）。血清中和実験では、同時にこれらの分離物が２つの最も一般的に用いられる一次分離物中和ＭＡｂ（２Ｆ５と２Ｇ１２）により中和される感受性を評価した（表２）。
【００５２】
【表２】

【００５３】
数値は、修飾ΔＶ２ｇｐ１４０（Ｊ４０８とＨ４４５）、非修飾ＳＦ１６２ｇｐ１４０（Ｐ６５５とＮ４７２）及び組換えｇｐ１２０（Ｌ７１４とＬ８１４）で免疫した動物から集めた血清（１：１０の希釈液）による一定のＨＩＶ−１分離物の中和％である。各分離物の共レセプターの使用を括弧に示す。中和％は材料及び方法に記載されているように計算され、免疫計画の開始前に同じ動物から集めた血清で記録された非特異的中和を考慮に入れた。^＆（Ａ）： Ｊ４０８とＨ４４５の動物のＤＮＡ＋タンパク質‘ブースター’免疫の２週間後に集めた血清及び（Ｂ）最後のタンパク質‘ブースター’免疫の２週間後に集めた血清。数値は、２〜３回の独立した実験からの平均である。２Ｆ５と２Ｇ１２による２５μｇ／ｍｌのＭＡｂでの中和に対するこれらの分離物の感受性も示されている。ＮＴ：評価せず。
サルにおけるＤＮＡ相の免疫化では異種分離物中和は記録されなかった（１：１０の希釈液で感染阻止５０％未満）。ＤＮＡ＋タンパク質‘ブースター’免疫の２週間後、修飾ΔＶ２ｇｐ１４０タンパク質でワクチン接種した２匹の動物から集めた血清は、試験した異種ＨＩＶ−１一次分離物の一部を中和した（図１０）。試験した最低血清希釈液（１：１０）において、また、免疫前血清で記録された非特異的中和を考慮に入れた場合（詳しくは材料及び方法を参照されたい）、８０〜９０％の感染阻止がＪ４０８によりＡＤＡ、９１ＵＳ０５６と９２ＵＳ７１４の分離物で、また、Ｈ４４５血清によりＡＤＡ、９２ＵＳ７１４と９２ＵＳ６６０の分離物でのみ記録された（図１０及び表２）。これらの２匹の動物から集めた血清の交差中和活性は異なった。例えば、９２ＵＳ６６０感染は、Ｈ４４５とＪ４０８の血清でそれぞれ８０％と５０％だけ阻止された。血清交差中和活性はその後の週の所見では低下した（図１０）。このＤＮＡ＋タンパク質‘ブースター’免疫の５週間後に集めた血清は、交差反応性中和活性がなかったが、ＳＦ１６２とＳＦ１６２ΔＶ２の分離物の強力な中和はなお記録された。
【００５４】
このＤＮＡ＋タンパク質‘ブースター’免疫後、非修飾免疫原でワクチン接種した動物における結合抗体力価は修飾免疫原でワクチン接種した動物より大きいという事実にもかかわらず（図８）、前者の血清は試験した異種分離物のいずれも中和することができなかった（表２）（即ち、５０％未満の特異的中和が記録された）。従って、ウサギにおいて非修飾免疫原は一部の異種ＨＩＶ−１一次分離物に対して中和抗体を惹起することができた（修飾免疫原より非常に効率が悪いが）（表１）が、アカゲザルにおいてはそのように惹起できなかった。
同時に、組換えＳＦ２由来ｇｐ１２０タンパク質で免疫したサルから集めた血清により中和される異種分離物の感受性を評価した。このタンパク質は、ワクチン候補として以前に評価されており、交差反応性中和抗体を惹起するのに無効であった。即ち、１：１０の血清希釈液で５０％未満の中和が記録された（Ｍａｓｃｏｌａ， Ｊ． Ｒ．， Ｓ． Ｗ． Ｓｎｙｄｅｒ， Ｏ． Ｓ． Ｗｅｉｓｌｏｗ， Ｓ． Ｍ． Ｂｅｌａｙ， Ｒ． Ｂ． Ｂｅｌｓｈｅ， Ｄ． Ｈ． Ｓｃｈｗａｒｔｚ， Ｍ． Ｌ． Ｃｌｅｍｅｎｔｓ， Ｒ． Ｄｏｌｉｎ， Ｂ． Ｓ． Ｇｒａｈａｍ， Ｇ． Ｊ． Ｇｏｒｓｅ， Ｍ． Ｃ． Ｋｅｅｆｅｒ， Ｍ． Ｊ． ＭｃＥｌｒａｔｈ， Ｍ． Ｃ． Ｗａｌｋｅｒ， Ｋ． Ｆ． Ｗａｇｎｅｒ， Ｊ． Ｇ． ＭｃＮｅｉｌ， Ｆ． Ｅ． ＭｃＣｕｔｃｈａｎ， Ｄ． Ｓ． Ｂｕｒｋｅ ＆ ｔｈｅ ＮＩＡＩＤ ＡＩＤＳ ｖａｃｃｉｎｅ ｅｖａｌｕａｔｉｏｎ ｇｒｏｕｐ． １９９６． Ｉｍｍｕｎｉｚａｔｉｏｎ ｗｉｔｈ ｅｎｖｅｌｏｐｅ ｓｕｂｕｎｉｔ ｖａｃｃｉｎｅ ｐｒｏｄｕｃｔｓ ｅｌｉｃｉｔｓ ｎｅｕｔｒａｌｉｚｉｎｇ ａｎｔｉｂｏｄｉｅｓ ａｇａｉｎｓｔ ｌａｂｏｒａｔｏｒｙ−ａｄａｐｔｅｄ ｂｕｔ ｎｏｔ ｐｒｉｍａｒｙ ｉｓｏｌａｔｅｓ ｏｆ ｈｕｍａｎ ｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙ ｖｉｒｕｓ ｔｙｐｅ １． Ｊ． Ｉｎｆｅｃｔ． Ｄｉｓ． １７３：３４０−３４８）。ここで試験した分離物はすべてＳＦ２ ｇｐ１２０タンパク質で惹起される抗体による中和に感受性でなかった（表２）。
【００５５】
修飾ΔＶ２ｇｐ１４０タンパク質による２回目の‘ブースター’免疫： 上記結果は修飾ΔＶ２ｇｐ１４０免疫原が非修飾免疫原より交差反応性中和抗体応答を惹起するのに実際に効果的であることを示したが、これらの応答は親ＳＦ１６２分離物に対して記録されたものより弱かった（図１１Ａ−Ｂ）。これらの応答の強さと幅を更に増大させる努力として、精製オリゴマーΔＶ２ｇｐ１４０タンパク質で１回更に免疫することによりＨ４４５とＪ４０８の動物において抗体力価を更に‘追加刺激’することが試みられた（このときＤＮＡ免疫はない）。
抗体力価の増大は実際にこのタンパク質‘追加刺激’後に記録されたので、ピーク値（１：１４５，０００終点ＥＬＩＳＡ力価）の力価はＤＮＡ＋タンパク質による最初の‘ブースター’免疫化で記録されたものより約３倍大きかった（図１１Ａ）。同時に、同種ＳＦ１６２ΔＶ２分離物と親ＳＦ１６２分離物に対する中和抗体の力価に著しい増加が見られた（図１１）。
この最後の‘追加刺激’の２週間と５週間後に集めた血清の中和可能性の差は報告されなかったが、結合抗体力価は同期間中に著しく減少した。しかしながら、予想外に、試験したほとんどの異種一次分離物に対する同じ血清の中和可能性がたいてい減少した（表２）。従って、ＢＺ１６７、９２ＵＳ６５７及びＡＤＡの分離物を除いて、試験した異種分離物のすべてが２回目の‘追加刺激’の２週間後に集めた血清による中和に抵抗した。面白いことに、分離物９２ＵＳ６５７は１回目の追加刺激後に集めた血清による中和に抵抗したが、２回目の追加刺激後に集めた血清による中和には感受性であった。
【００５６】
修飾ΔＶ２ｇｐ１４０でワクチン接種したアカゲザルにおける抗Ｖ３ループ抗体の産生： 親ＳＦ１６２ウイルスと同種ＳＦ１６２ΔＶ２ウイルスに対する中和活性の増大と２回目の‘ブースター’免疫後の異種分離物に対する中和活性の減少の説明は、修飾ΔＶ２ｇｐ１４０タンパク質による多重免疫化がＳＦ１６２エンベロープとＳＦ１６２ΔＶ２エンベロープ上でユニークに（又は優先して）発現するエピトープに対する抗体の力価を増大させるということである。ΔＶ２ｇｐ１４０による多重免疫化により高力価の抗Ｖ３ループ抗体の産生が得られることはありそうなことである。そのような抗体の力価を求めるために、ＳＦ１６２／ＳＦ１６２ΔＶ２由来Ｖ３ループを用いたＶ３ループぺプチドに基づくＥＬＩＳＡ分析を用いた（図１２Ａ−Ｂ）。このぺプチドは、線状（４４７Ｄ）エピトープ（Ｃｏｎｌｅｙ， Ａ． Ｊ．， Ｍ． Ｋ． Ｇｏｒｎｙ， Ｊ． Ａ． Ｋｅｓｓｌｅｒ， ｓｅｃｏｎｄ， Ｌ． Ｊ． Ｂｏｏｔｓ， Ｍ． Ｏｓｓｏｒｉｏ−Ｃａｓｔｒｏ， Ｓ． Ｋｏｅｎｉｇ， Ｄ． Ｗ． Ｌｉｎｅｂｅｒｇｅｒ， Ｅ． Ａ． Ｅｍｉｎｉ， Ｃ． Ｗｉｌｌｉａｍｓ ＆ Ｓ． Ｚｏｌｌａ−Ｐａｚｎｅｒ． １９９４． Ｎｅｕｔｒａｌｉｚａｔｉｏｎ ｏｆ ｐｒｉｍａｒｙ ｈｕｍａｎ ｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙ ｖｉｒｕｓ ｔｙｐｅ １ ｉｓｏｌａｔｅｓ ｂｙ ｔｈｅ ｂｒｏａｄｌｙ ｒｅａｃｔｉｖｅ ａｎｔｉ−Ｖ３ ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｙ， ４４７−５２Ｄ． Ｊ． Ｖｉｒｏｌ． ６８：６９９４−７０００； Ｇｏｒｎｙ， Ｍ． Ｋ．， Ａ． Ｊ． Ｃｏｎｌｅｙ， Ｓ． Ｋａｒｗｏｗｓｋａ， Ａ． Ｂｕｃｈｂｉｎｄｅｒ， Ｊ．−Ｙ． Ｘｕ， Ｅ． Ａ． Ｅｍｉｎｉ， Ｓ． Ｋｏｅｎｉｇ ＆ Ｓ． Ｚｏｌｌａ−Ｐａｚｎｅｒ． １９９２． Ｎｅｕｔｒａｌｉｚａｔｉｏｎ ｏｆ ｄｉｖｅｒｓｅ ｈｕｍａｎ ｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙ ｖｉｒｕｓ ｔｙｐｅ １ ｖａｒｉａｎｔｓ ｂｙ ａｎ ａｎｔｉ−Ｖ３ ｈｕｍａｎ ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｙ． Ｊ． Ｖｉｒｏｌ． ６６：７５３８−７５４２）にもコンフォメーショナル（３９１〜９５Ｄ）エピトープ（Ｓｅｌｉｇｍａｎ， Ｓ． Ｊ．， Ｊ． Ｍ． Ｂｉｎｌｅｙ， Ｍ． Ｋ． Ｇｏｒｎｙ， Ｄ． Ｒ． Ｂｕｒｔｏｎ， Ｓ． Ｚｏｌｌａ−Ｐａｚｎｅｒ ＆ Ｋ． Ａ． Ｓｏｋｏｌｏｗｓｋｉ． １９９６． Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｂｙ ｓｅｒｉａｌ ｃｏｍｐｅｔｉｔｉｏｎ ＥＬＩＳＡｓ ｏｆ ＨＩＶ−１ Ｖ３ ｌｏｏｐ ｅｐｉｔｏｐｅｓ ｒｅｃｏｇｎｉｚｅｄ ｂｙ ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｉｅｓ． Ｍｏｌ． Ｉｍｍｕｎｏｌ． ３３：７３７−７４５）にも結合する抗体によって認識された（図１１Ａ）。
【００５７】
抗Ｖ３ループ抗体は修飾ΔＶ２ｇｐ１４０免疫原によるサルの免疫化時に産生されたが、力価は全体のエンベロープに対するものより非常に小さかった（図１１Ｂ）。更に、２回目の‘ブースター’免疫は抗Ｖ３ループ抗体の力価を高めなかった。しかしながら、これらの動物の血清中に存在するある種の抗Ｖ３ループ抗体がＥＬＩＳＡ方式でＶ３ループぺプチドと効率よく相互作用することができないが、未変性エンベロープ上のエピトープに結合することができることは留意すべきである（Ｍｏｏｒｅ， Ｊ． Ｐ． １９９３． Ｔｈｅ ｒｅａｃｔｉｖｉｔｉｅｓ ｏｆ ＨＩＶ−１＋ｈｕｍａｎ ｓｅｒａ ｗｉｔｈ ｓｏｌｉｄ−ｐｈａｓｅ Ｖ３ ｌｏｏｐ ｐｅｐｔｉｄｅｓ ｃａｎ ｂｅ ｐｏｏｒ ｐｒｅｄｉｃｔｏｒｓ ｏｆ ｔｈｅｉｒ ｒｅａｃｔｉｖｉｔｉｅｓ ｗｉｔｈ Ｖ３ ｌｏｏｐｓ ｏｎ ｎａｔｉｖｅ ｇｐ １２０ ｍｏｌｅｃｕｌｅｓ． ＡＩＤＳ Ｒｅｓ． Ｈｕｍ． Ｒｅｔｒｏｖｉｒｕｓｅｓ ９：２０９−１９）。更に、ここで用いられるＶ３ループぺプチドはＶ３ループのカルボキシ末端とアミノ末端にかからず、分析はこれらの２つの領域をターゲッティングする抗体を検出しない。従って、修飾ΔＶ２ｇｐ１４０で惹起する抗体のエピトープ特異性を更に詳しく調べることが必要である。
【００５８】
図１３Ａ及び１３Ｂは、異なるクレードのＨＩＶ−１ウイルスを中和することによる本発明の免疫原によって惹起される異種免疫応答を示すグラフである。図１３ＡはＨ４４５の動物からの血清を用いた中和を示し、図１３ＢはＪ４０８からの血清を用いた中和を示している。
本発明を個々の実施態様、個々の材料、手順及び実施例によって記述し具体的に説明してきたが、本発明が材料の具体的な材料の組合わせやそのために選ばれた手順に限定されないことは理解される。実際は、本明細書に記載されたもおのほかに本発明の種々の変更が上記説明と添付の図面から当業者には明らかである。そのような変更は、前述の特許請求の範囲の範囲内に包含されるものである。
種々の文献が本明細書に引用され、その開示は本願明細書に含まれるものとする。
【図面の簡単な説明】
【図１】
免疫化で抗ＨＩＶ−１エンベロープ結合抗体の産生を示すグラフである。ワクチン、即ち、精製ＳＦ１６２ΔＶ２ ｇｐ１４０オリゴマータンパク質に対して、免疫計画全体で動物Ｊ４０８とＨ４４５における結合抗体のエンベロープ特異的力価を求めた。点線は、免疫化の時間を示し、矢印はウイルス攻撃の時間を示している。
【図２】
ＨＩＶ−１中和抗体の産生を示すグラフである。同種ＳＦ１６２ΔＶ２ウイルスや親ＳＦ１６２ウイルスに対する中和抗体の存在を、免疫計画中の様々な時点で求めた。○： 出血前； ▲： ３回目のＤＮＡ免疫化の１ヶ月後； ■： １回目の‘追加免疫’の２週間後； 及び◆： ２回目の‘追加免疫’の２週間後。
【図３】
ＣＤ８＋ Ｔリンパ球の枯渇： ＣＤ８＋ Ｔリンパ球は抗ＣＤ８ ＭＡｂ ＯＫＴ８Ｆのボーラス注射（矢印）によりワクチン接種した動物から枯渇したことを示すグラフである。ＳＨＩＶ １６２Ｐ４攻撃（点線）前後の種々の時点で集めた試料において、ワクチン接種した動物とワクチン接種していない動物からの循環しているＣＤ４＋（黒い丸）、ＣＤ８＋ Ｔ（白い丸）及び全ＣＤ３＋ Ｔリンパ球（星印）の数を求めた。
【図４Ａ−Ｂ】
ＳＨＩＶ１６２Ｐ４曝露後のウイルス負荷と抗ＨＩＶエンベロープ抗体力価の産生を示すグラフである。（Ａ）ウイルス負荷は血漿１ ｍｌ当たりのＲＮＡコピー数として示されている。点線は、この分析の検出限界を示している（＜５００コピー／ｍｌ）。十字：ワクチン接種していない動物ＡＴ５４を、攻撃後のサルエイズ（ＳＡＩＤＳ）の発症の１１１日後に安楽死させた。矢印は、ワクチン接種した動物の末梢にＣＤ８＋細胞が再び現れた時点を示している。（Ｂ）ＳＨＩＶ１６２Ｐ４攻撃後の抗ＨＩＶエンベロープ抗体の産生をＳＦ１６２ΔＶ２ ｇｐ１４０に基づくＥＬＩＳＡ法によりモニターした。終点ＥＬＩＳＡ力価が示されている。
【図５】
ワクチン接種したサルとワクチン接種していないサルにおけるＳＩＶ−ｇａｇ／ｐｏｌ抗原とＨＩＶ ｅｎｖ抗原への動物の血清変換を示す図である。
【図６】
ウサギにおける抗体産生を示す図である。抗エンベロープ抗体の産生をＥＬＩＳＡ法により求めた。６匹の動物（Ａ１〜Ａ６）を非修飾ＳＦ１６２ｇｐ１４０免疫原を発現するＤＮＡで、６匹（Ａ７〜Ａ１２）を修飾ΔＶ２ｇｐ１４０免疫原を発現するＤＮＡで免疫化した。それぞれの免疫化の２週間後に、ＳＦ１６２ｇｐ１４０とΔＶ２ｇｐ１４０のオリゴマータンパク質を用いたＥＬＩＳＡ法により力価を求めた。点線は各免疫化の時点を示している。
【図７ａ】
ウサギ血清によるＳＦ１６２ΔＶ２ウイルスとＳＦ１６２ウイルスの中和を示すグラフである。ＳＦ１６２ΔＶ２（Ａ）ウイルスとＳＦ１６２（Ｂ）ウイルスに対する３回目と５回目の免疫化後に集めた血清を用いた中和実験からの結果が示されている。データは少なくとも３回の独立した実験の代表である。記号は３回の実験ウェルからの平均中和％と標準偏差を示している。点線は感染の５０％阻止、７０％阻止及び９０％阻止を示している。点線と星印（対照）はＤＮＡベクターのみで免疫化された動物から集めた血清で得られた中和曲線であり、非特異的中和を示している。
【図７Ｂ】
ウサギ血清によるＳＦ１６２ΔＶ２ウイルスとＳＦ１６２ウイルスの中和を示すグラフである。ＳＦ１６２ΔＶ２（Ａ）ウイルスとＳＦ１６２（Ｂ）ウイルスに対する３回目と５回目の免疫化後に集めた血清を用いた中和実験からの結果が示されている。データは少なくとも３回の独立した実験の代表である。記号は３回の実験ウェルからの平均中和％と標準偏差を示している。点線は感染の５０％阻止、７０％阻止及び９０％阻止を示している。点線と星印（対照）はＤＮＡベクターのみで免疫化された動物から集めた血清で得られた中和曲線であり、非特異的中和を示している。
【図８】
アカゲザルにおける抗体の産生を示すグラフである。修飾ΔＶ２ｇｐ１４０免疫原で免疫化した動物（Ｊ４０８とＨ４４５）及び非修飾ＳＦ１６２ｇｐ１４０免疫原で免疫化した２種の動物（Ｐ６５５とＮ４７２）、並びにＤＮＡベクターのみで免疫化した対照動物（Ｍ８４４とＨ４７３）における抗エンベロープ抗体の産生を対応するタンパク質を用いたＥＬＩＳＡ法により求めた。点線は免疫化の時点を示している。ＤＮＡ： 動物は各免疫原のｇｐ１４０型を発現するＤＮＡベクターで３回１ヶ月毎に免疫処置した。対照動物はＤＮＡベクターのみを投与した。ＤＮＡ＋タンパク質： 動物は４回免疫処置し、同時にＭＦ−５９Ｃのアジュバントを用いた対応するＣＨＯ産生ｇｐ１４０オリゴマータンパク質で免疫した。対照動物はアジュバントのみを投与した。
【図９Ａ】
アカゲザル血清の中和活性を示すグラフである。修飾ΔＶ２ｇｐ１４０（Ａ）と非修飾（Ｂ）ＳＦ１６２ｇｐ１４０免疫原で免疫した動物から集めた血清のＳＦ１６２ウイルスとＳＦ１６２ΔＶ２ウイルスに対する中和活性を実施例２に記載されたように求めた。点線は、感染の５０％阻止、７０％阻止及び９０％阻止を示している。結果は３〜５回の独立した実験の代表である。データは、３回の実験のウェルからの平均と標準偏差を示している。出血前（プレブリード（Ｐｒｅ−ｂｌｅｅｄ））： ワクチン接種の開始前に集めた血清； ２回目のＤＮＡ及び３回目のＤＮＡ： それぞれ２回目と３回目のＤＮＡ投与の１ヶ月後に集めた血清； 追加刺激の２週間後と４週間後： それぞれＤＮＡ＋タンパク質‘ブースター’免疫の２週間後と４週間後に集めた血清。
【図９Ｂ】
アカゲザル血清の中和活性を示すグラフである。修飾ΔＶ２ｇｐ１４０（Ａ）と非修飾（Ｂ）ＳＦ１６２ｇｐ１４０免疫原で免疫した動物から集めた血清のＳＦ１６２ウイルスとＳＦ１６２ΔＶ２ウイルスに対する中和活性を実施例２に記載されたように求めた。点線は、感染の５０％阻止、７０％阻止及び９０％阻止を示している。結果は３〜５回の独立した実験の代表である。データは、３回の実験のウェルからの平均と標準偏差を示している。出血前（プレブリード（Ｐｒｅ−ｂｌｅｅｄ））： ワクチン接種の開始前に集めた血清； ２回目のＤＮＡ及び３回目のＤＮＡ： それぞれ２回目と３回目のＤＮＡ投与の１ヶ月後に集めた血清； 追加刺激の２週間後と４週間後： それぞれＤＮＡ＋タンパク質‘ブースター’免疫の２週間後と４週間後に集めた血清。
【図１０】
サル血清による異種クレードＢ ＨＩＶ−１一次分離物の中和を示すグラフである。ワクチンに異種ＨＩＶ−１一次分離物に対する、ＤＮＡ＋タンパク質‘ブースター’免疫の２週間後と４週間後に集めた血清の中和活性を実施例２に記載されたように求めた。点線は、感染の５０％阻止、７０％阻止及び９０％阻止を示している。数値は特異的中和を示し、ワクチン接種後に集めた血清で記録されたウイルス中和％とワクチン接種開始前に集めた血清で記録されたウイルス中和％との間の差として定義される。データ点は、２回の独立した実験からの平均特異的中和％を示している。
【図１１Ａ】
修飾ΔＶ２ｇｐ１４０タンパク質による２回目の‘ブースター’免疫後の結合抗体と中和抗体の産生を示すグラフである。（Ａ）修飾ΔＶ２ｇｐ１４０でワクチン接種した２種のアカゲザル（Ｊ４０８とＨ４４５）における抗エンベロープ抗体の産生を、実施例２に記載されたようにＥＬＩＳＡ法により求めた。点線は免疫化時を示している。ＤＮＡ： その免疫原のｇｐ１４０型を発現するＤＮＡベクターで３回１ヶ月毎に免疫処置した。ＤＮＡ＋タンパク質： 動物は４回目のＤＮＡ免疫処置と精製ΔＶ２ｇｐ１４０オリゴマータンパク質を投与した。タンパク質： 動物を精製ΔＶ２ｇｐ１４０オリゴマータンパク質のみで免疫した。（Ｂ）２回目の‘追加刺激’後の血清のＳＦ１６２ΔＶ２分離物とＳＦ１６２分離物に対する中和活性を１回目の‘追加刺激’後に集めた血清と比較した（図４を参照されたい）。免疫前血清（出血前（プレブリード））で記録された非特異的中和も示されている。
【図１１Ｂ】
修飾ΔＶ２ｇｐ１４０タンパク質による２回目の‘ブースター’免疫後の結合抗体と中和抗体の産生を示すグラフである。（Ａ）修飾ΔＶ２ｇｐ１４０でワクチン接種した２種のアカゲザル（Ｊ４０８とＨ４４５）における抗エンベロープ抗体の産生を、実施例２に記載されたようにＥＬＩＳＡ法により求めた。点線は免疫化時を示している。ＤＮＡ： その免疫原のｇｐ１４０型を発現するＤＮＡベクターで３回１ヶ月毎に免疫処置した。ＤＮＡ＋タンパク質： 動物は４回目のＤＮＡ免疫処置と精製ΔＶ２ｇｐ１４０オリゴマータンパク質を投与した。タンパク質： 動物を精製ΔＶ２ｇｐ１４０オリゴマータンパク質のみで免疫した。（Ｂ）２回目の‘追加刺激’後の血清のＳＦ１６２ΔＶ２分離物とＳＦ１６２分離物に対する中和活性を１回目の‘追加刺激’後に集めた血清と比較した（図４を参照されたい）。免疫前血清（出血前（プレブリード））で記録された非特異的中和も示されている。
【図１２Ａ−Ｂ】
修飾ΔＶ２ｇｐ１４０免疫原で免疫したサル（マカク）から集めた血清における抗Ｖ３ループ抗体の存在を示すグラフである。ＳＦ１６２／ＳＦ１６２ΔＶ２エンベロープ由来のＶ３ループぺプチドを用いたＥＬＩＳＡ法の使用により抗Ｖ３ループ抗体の産生を求めた。（Ａ）まず、捕捉Ｖ３ループぺプチドが線状（４４７Ｄ）Ｖ３エピトープとコンフォメーショナル（３９１−９５Ｄ）Ｖ３ループエピトープを認識する特異的抗Ｖ３ループＭＡｂと相互作用するかを調べた。（Ｂ）次に、２種のワクチン接種した動物から１回目と２回目の追加刺激の２週間後と４週間後に集めた血清中に存在する抗Ｖ３ループ抗体の力価を求めた。比較として同じ血清中に存在する全抗エンベロープ抗体の力価も含めた。
【図１３Ａ】
Ｖ２領域欠失があるＨＩＶ−１クレードＢ免疫原由来修飾エンベロープタンパク質で免疫した２種の動物からの血清によるクレードＡ、Ｅ及びＤのＨＩＶ−１の中和を示すグラフである。
【図１３Ｂ】
Ｖ２領域欠失があるＨＩＶ−１クレードＢ免疫原由来修飾エンベロープタンパク質で免疫した２種の動物からの血清によるクレードＡ、Ｅ及びＤのＨＩＶ−１の中和を示すグラフである。
【図１４】
全長ＳＦ１６２ΔＶ２ ｇｐ１４０エンベロープタンパク質のポリヌクレオチド配列を示すアミノ酸配列である（配列番号１）。
【図１５】
ＳＦ１６２ΔＶ２ ｇｐ１４０エンベロープタンパク質断片のポリヌクレオチド配列を示すアミノ酸配列である（配列番号３）。
【図１６】
全長ＳＦ １６２ΔＶ２ ｇｐ１４０エンベロープタンパク質を示すアミノ酸配列である（配列番号２）。
【図１７】
ＳＦ１６２ΔＶ２ ｇｐ１４０エンベロープタンパク質断片を示すアミノ酸配列である（配列番号４）。[0001]
Background of the Invention
DNA immunization stimulates both the cellular and humoral arms of the immune system (Liu, MA, Y. Yasutomi, ME-Davis, HC Perry, DC). Freed, N.L.Letvin & J.W. Shiver. 1996. Vaccination of mice and nonhuman primates using HIV-gene-gun-containing DNA, vol.48, Kargersger. E. Davies, H. C. Perry, D. C. Freed, & M. A. Liu. 1996. Human and cellular immunities elicited by HIV-1 DNA vac. 85. 1317-1324; Shiver, JW, H. C. Perry, M.-E. Davies, D.C. Freed & M.A. Liu. 1995. Cytotoxicic T. ination.J. Pharm.Sci. Lymphocite and helper T cell responses following HIV HIV Nucleotide vaccination. DNA Vaccines. 772: 198-208, J. Elm, J.U., J.U., J.U. Immunities elicited by DNA vaccines: Application to the human Immunodeficiency virus and influenza. Adv. Drug Del. Rev. 21: 19-31-18) together with a slowly replicating virus such as HIV-1 in chimpanzees elicit an immune response that can prevent infection of animals (Boyer, J. D., KEE Ugen, B. Wang, M. Agadjanyan, L. Gilbet, M.L. Bagarazzzi, M. Chattergoon, P. Frost, A. J. avadian, W.V.V. Refaeli, R .; B. Ciccarelli, D.C. McCallus, L .; Coney & D. B. Weiner. 1997. Protection of Chinas from high-dose heterologous HIV-1 challenge by DNA vaccination. Nature Med. 3: 526-532).
[0002]
However, if the challenge virus replicates efficiently in the host, such as SIV or SHIV in monkeys, the DNA-evoked immune response provides only partial protection (Boyer, JD, B. Wang, KE Ugen, M. Agadjanyan, A. Javadian, P. Frost, K. Dang, R. A. Carrano, R. Ciccarelli, L. Conney, W.V. Williams & D.B.viet.vivi. Lu, S., J. Immun responses in non-human primates through DNA immunization. J. Med. Protomol. 25: 242-250; rthos, DC Montefiori, Y. Yasutomi, K. Manson, F. Mustafa, E. Johnson, J.C. Santoro, J. Wissink, J.I.N.R.J.M.R. Letvin, M. Wyand & HL Robinson, 1996. Simian immunodeficiency virus DNA vaccine trial in macaques. J. Virol, 70: 3978-Ron, Rob. Johnson, KL Manson, ML Kalish, JD Lifeson, TA Rizvi, S. Lu, S. L. Hu, GP Mazzara, DL Panicali, JG Herdon, R. Glickman, MA Candido, SL Lydy, MS Wyand & H. M. McClure. Neutralizing antibody-independent involvement of immunodeficiency virus challenges by DNA priming and recombinant pox virus bots. Nature Medicine. 5: 526-34).
[0003]
In order to enhance the strength of these responses, especially the production of high anti-HIV / SIV envelope antibody titers, continuous administration of a soluble viral envelope protein, virus particle or recombinant vaccinia virus expressing the HIV / SIV envelope is required. (Agadjyanan, MG, NN Trivedi, S. Kudchodkar, M. Bennett, W. Levine, A. Lin, J. Boyer, D.J.J.E.G., UK, K.E. & DB Weiner 1997. An HIV type 2 DNA vaccinates cross-reactive immune response against HIV type 2 and SIV.AIDS Res. 13: 1561-1572; Barnett, SW, JM Klinger, B. Doe, C. M. Walker, L. Hansen, A. M. Duliege & F. M. Singl. Boost Immunization Strategies against HIV. AIDS Res. Hum. Retroviruses. 14 Suppl 3: S299-309; Letvin, N.L., D.C., Montyori, Y.M.Y.Y.M. Davies, C. Lekutis, M. Alloy, DL Freed, CI Lord, LKK Handdt, MA Liu & JW ...... Shiver 1997. Potent protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination Proc Natl Acad Sci 94: 9378-9383;
[0004]
Richmond, J. et al. F. , S.M. Lu, J.M. C. Santro, J .; Weng, S.W. L. Hu, D.S. C. Montefiori & H.M. L. Robinson. 1998. Studies of the Neutralizing Activity and Ability of Anti-Human Immunodeficiency Virus Type 1 Environment of the Organized Bibliography of the United States 72: 9092-100; Richmond, J. M .; F. L. , Mustafa, S.M. Lu, J.M. C. Santro, J .; Weng, M.W. O'Connell, E .; M. Fenyo, J .; L. Hurwitz, D.S. C. Montefiori & H.M. L. Robinson. 1997. Screening of HIV-1 Env glycoproteins for the availability to raise neutralizing antibody using DNA immu nization and recombinant vaccina virus. Virology. 230: 265-274; Robinson, H .; L. , D. C. Montefiori, R .; P. Johnson, K.C. H. Manson, M .; L. Kalish, J .; D. Lifson, T .; A. Rizvi, S.M. Lu. Hu, G. P. Mazzara, D.M. L. Panicali, J .; G. FIG. Herndon, R .; Glickman, M .; A. Candido, S.M. L. Lydy, M .; S. Wyand & H. M. McClure, 1999. Neutralizing antibody-independent intervention of immunodeficiency viruses challenges by DNA priming and recombinant pox virus botany. Nature Medicine. 5: 526-34).
[0005]
This bimodal immunization elicits a response that can protect rhesus monkeys (Rh) from infection with SHIV (Letvin, NL, DC Montefiori, Y. Yasutomi, HC Perry, M.-E. Davies, C. Lekutis, M. Alloy, D.L. Freeed, C.I.L.L.D., L.K. Handdt, M.A. Liu & J.W. Shiv. Responsible investment proposals. generated by bimodal HIV envelope DNA plus protein vaccination. Proc. Natl. Acad. Sci. 94: 9378-9383; Robinson, H .; L., DC Montefiori, RP Johnson, KH Manson, ML Kalish, JD Lifeson, T. A. Rizvi, S. Lu, S. L. Hu, G. P. Mazzara, DL Panicali, J. G. Herndon, R. Glickman, MA Candido, SL. Lydy, MS. Wyand & H. M. memoy. Independent Containment of Immunodeficiency Virus Challenges by DNA Priming and Recombinant Pox Virus Booster Booster Immunizations. Nature Medicine. 5: 526-34). However, since the vaccination method produced both a cellular and a humoral antiviral response, the recorded protection was induced by DNA immunization in a cellular and / or humoral antiviral response. It is not clear if they were brokered by. By assessing and comparing the role of each of these two types of responses in antiviral protection, more effective DNA immunization protocols can be developed.
[0006]
Analysis of the crystal structure of the gp120 HIV envelope subunit shows that the neutralizing epitope is mainly clustered on one surface of the protein that is naturally closed in the oligomer envelope form, ie, the surface of virions and infected cells. (Kwong, PD, RD Wyatt, J. Robinson, RW Sweet, J. Sodroski & W. A. Hendrickson. 1998. Structure of HIVA. envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature (London) 393: 648-659; Wyatt, R., P, D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, WA Hendrickeson & J. G. Socitechnique, 1998, Therapeuticgene. (London) 393: 705-711).
[0007]
These structural findings have been supported by numerous immunochemical and virological experiments (Bou-Habib, DC, G. roderiquez, T. Oravesz, P.W. Berman, P. Lusso). & M. A. Norcross 1994. Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization J. Virol 68:... 6006-6013; Moore, J. P., J. A. McKeating , Y. Huang, A. Askenazi & D. D. Ho. 1 992. Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates J. Virol 66:.. 235-243; Reitter, J. N., RE Mcans & RC Desrosiers. 1998. A role for carbohydrates in immune evolution in AIDS.Nat.Med. 4: 679-684; Satentau, & Q.J. P. Moore 1991. Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding J. Exp Med 174:.... 407-415; Sattentau, Q. J., J. P. Moore, F. Vignaux, 1993. F. Traincard & P. Poignard.1993.Conformal changes induced in the environment glycoproteins of the human and simulating disease rebirth reciprocity. g. J. Virol. 67: 7383-7393;
[0008]
Stamatatos, L .; & C. Cheng-Mayer. 1995. Structual modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding. J. Virol. 69: 6191-6198; Sullivan, N .; Y. Sun, J.M. Li, W.S. Hofmann & J.A. Sodroski. 1995. Replicative function and neutralization sensibility of envelope glycoproteins from primary and T-cell line-passed humidifice. J. Virol. 69: 4413-4422; Wyatt, R .; , J. et al. Moore, M .; Accola, E .; Desjardin, J.M. Robinson & J.S. Sodroski. 1995. Involvement of the V1 / V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epidep. J. Virol. 69: 5723-5733; Wyatt, R .; , N .; Sullivan, M .; Tali, H .; Repke, D .; Ho, J .; Robinson, M .; Posner & J.S. Sodroski. 1993. Functional and immunological charactrization of human immunodeficiency virus type 1 envelope glycoproteins containing removals of the reimaging. J. Virol. 67: 4557-4565).
The present invention relates to the enhancement of effective vaccination against HIV-1.
Citation of a document herein shall not be construed as an admission that such document can be used as prior art to the present invention.
[0009]
Summary of the Invention
According to the present invention, there is provided a method for raising a heterologous immune response against HIV-1 in an animal, the animal comprising at least one modified HIV-1 envelope protein or a fragment thereof, or at least one modified HIV-1 The method is provided by immunizing with a DNA or virus encoding the envelope protein or a fragment thereof, or a combination thereof, wherein the modified envelope protein has a deletion of the HIV-1 envelope protein V2 region. The modified HIV-1 envelope protein may be a recombinant protein of a fragment expressed in a mammalian cell. Preferably, the modified HIV-1 envelope protein or a fragment thereof is glycosylated. The immunized animal has shown an immune response to at least one strain of HIV-1 other than the immunogen. In a preferred embodiment, the immune response comprises a humoral response. In a further preferred embodiment, the humoral response includes a neutralizing antibody, and in a most preferred embodiment, a protective antibody. Preferably, the animal is a human.
[0010]
In a non-limiting example, the immunogen is a modified HIV-1 envelope protein from Clade B HIV-1 strain or a fragment thereof, or a DNA encoding a modified HIV-1 envelope protein from Clade B HIV-1 strain or Contains a virus or a fragment thereof. In a preferred embodiment, the HIV-1 strain is SF162. As an example, the modified HIV-1 envelope protein or fragment thereof is SEQ ID NO: 2 or SEQ ID NO: 4, and the DNA encoding at least one modified HIV-1 envelope protein or fragment thereof is SEQ ID NO: 1 or SEQ ID NO: 4. 3.
[0011]
In another broad aspect of the invention, there is provided at least one modified HIV-1 envelope protein or fragment thereof, wherein the HIV-1 envelope protein has a V2 region deletion, to immunize the animal against the HIV-1 virus. Or a DNA or virus encoding at least one of the modified HIV-1 envelope proteins or fragments thereof, or a combination thereof, in an effective amount for inducing a heterologous immune response, and a pharmaceutically acceptable carrier or excipient. A vaccine pharmaceutical composition is provided. Modified HIV-1 envelope proteins or fragments thereof can be expressed in mammalian cells. It may be glycosylated. In embodiments, the modified HIV-1 envelope protein or fragment thereof is from a Clade B HIV-1 strain. In a preferred embodiment, the HIV-1 strain is SF162. As a non-limiting example, the modified HIV-1 envelope protein or fragment thereof is SEQ ID NO: 2 or SEQ ID NO: 4, and the DNA encoding at least one of the modified HIV-1 envelope proteins or fragments thereof is SEQ ID NO: 1. Or, it is SEQ ID NO: 3. Immunization or vaccination of an animal with the aforementioned vaccine pharmaceutical composition elicits a heterologous immune response against HIV-1. The response includes a humoral response. In embodiments, the humoral response includes a neutralizing antibody. In a preferred embodiment, the raised antibodies are protective antibodies.
[0012]
The present invention also provides a method of evaluating whether a compound is capable of producing at least one neutralizing antibody against at least one heterologous HIV-1 strain in an animal, comprising immunizing the animal with the compound. Depleting the animal's CD8 + cells, and screening the animal for the presence of neutralizing antibodies, preferably protective antibodies, against at least one heterologous HIV-1 strain. In a preferred embodiment, the step of depleting is performed by administering an anti-CD8 monoclonal antibody to said animal. The compound may be an HIV-1 derived polypeptide or a fragment thereof or a DNA or virus encoding the peptide or a fragment thereof, wherein the immunogen is a virus or DNA vaccine, a protein, or a combination thereof. Contains. The protective antibody is preferably a neutralizing antibody, most preferably a protective antibody. The animal for detecting the protective antibody is a wild-type HIV-1 or SHIV strain, or an animal capable of responding to the protective antibody against wild-type HIV-1 or SHIV-1 and infectable.
The present invention further relates to a method for producing the above-mentioned protein, protein fragment, DNA or virus encoding the protein or protein fragment. Preferably, the protein immunogen is expressed in mammalian cells and is therefore glycosylated.
These and other aspects of the present invention will be better understood with the following drawings and detailed description.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
We have shown that strong neutralization as part of an anti-HIV-1 envelope-specific immune response by animal immunization with a modified HIV-1 envelope protein having a deletion in the V2 (secondary hypervariable) region. We have made the surprising discovery that antibodies are raised. Furthermore, the immune response is not only to the wild-type of the immunogen envelope protein, but also to other HIV-1 viruses in and out of the clade from which the immunogen is derived. This strong heterologous immune response, especially a strong humoral response, offers a new means of vaccination for prevention and treatment of HIV infection, among other immunotherapies. The present invention relates to both DNA vaccines, viral vaccines or protein vaccines comprising one or more envelope proteins or fragments thereof with a deletion in the V2 region, and methods of using the same.
[0014]
In a non-limiting embodiment, immunization can be performed with DNA or a virus encoding an HIV-1 envelope protein or a fragment thereof that has a deletion in the V2 region. As described in the Examples below, a DNA vector capable of expressing a modified gp140 envelope protein from HIV-1 strain SF162 (clade B) containing a partial deletion in the V2 hypervariable region was prepared. In this case, the first 27 N-terminal amino acids (81 nucleotides) of the DNA and protein sequences, respectively, were not expressed. These DNA and protein fragments of the modified gp140 of SF162 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The corresponding full-length sequence SEQ ID NO: 1 and SEQ ID NO: 2 are used for the same purpose. DNA immunization of monkeys elicited an immune response containing strong neutralizing antibodies. When CD8 + T lymphocytes were depleted and challenged with SHIV162P4, vaccinated animals had lower viremia peaks, faster virus clearance from plasma, and slower seroconversion compared to unimmunized control animals. . These results demonstrate that the immunogens of the present invention elicit a strong protective humoral response. In addition, as noted above, cross-neutralizing reactivity was observed against several heterologous HIV-1 strains, supporting the utility of V2-deleted immunogens in raising a universal immune response against HIV-1 strains. Was. In immunized rabbits, the modified (V2-deleted) immunogen is even more effective in raising neutralizing antibodies against the homologous parent SF162 virus and also against some heterologous HIV-1 isolates. In monkeys, the modified immunogen was able to elicit neutralizing antibodies only against heterologous isolates.
[0015]
The present invention relates to immunization types or protocols, such as DNA, viruses, proteins, combinations thereof, or at least one of the immunogens described herein, plus one or more adjuvants or sets of materials. The type or protocol of immunization using the combination, and HIV- containing a deletion in the V2 (secondary hypervariable) loop (also referred to herein as the V2 domain or V2 region) as an immunogen. (1) An immunization protocol using a protein or DNA encoding a virus envelope protein. The wild-type sequence of the HIV-1 envelope protein candidate for deletion in the V2 region in the protein, DNA or viral immunogens described herein can be found at http: // idiotype. lanl. gov /, and all such sequences are incorporated herein in their entirety as starting sequences for the preparation of immunogens. One or a combination of such immunogens can be used together. In addition, various other modifications of the modified (ie, containing the V2 loop deletion) envelope protein of the invention or the DNA encoding the modified envelope protein of the invention can be made without departing from the invention. For example, the DNA or viral nucleotide sequence encoding the native envelope writer of the modified protein is replaced with a signal peptide of a human tissue-specific plasminogen activator gene for high protein expression in mammalian cells, for example. be able to.
[0016]
Other signal peptides can also be used. In other embodiments, a portion of the modified protein or its encoding DNA sequence may be truncated to obtain an immunogen to produce a neutralized humoral response, and such modifications are not fully described herein. included. Preferably, the fragment is truncated at the N-terminus of the modified protein or the DNA or virus encoding the modified protein, wherein the truncation is from 1 to about 30 amino acids, but is not so limited, and as used herein. Other truncations that provide an immunogen with the described immunological properties are also included. In addition, expression of the DNA construct in mammalian cells as shown in the examples herein results in glycosylated proteins glycosylated at asparagine residues as shown in FIGS. 16 and 17. The resulting protein immunogenic compositions also include glycosylated forms of the protein. Thus, the aforementioned non-limiting examples of immunogenic aspects of the proteins and DNAs of the invention that commonly include a deletion in the V2 loop domain include the phrase modified protein or fragment thereof, or the modified protein or fragment thereof. DNA or virus.
[0017]
The V2 domain is one of the hypervariable regions of the gp120 subunit of the HIV envelope. The length (number of amino acids) and degree of glycosylation varies between HIV isolates. In the case of the SF162 virus, the V2 loop contains 40 amino acids. In the experiments of the present invention, 30 amino acids were removed from the central region of the V2 loop and replaced with GAG peptides. One skilled in the art can make other deletions in the V2 domain of this strain, or deletions in the V2 region of other strains, which will retain the same immune response eliciting properties without departing from the scope and spirit of the invention. As shown, such characteristics can be easily evaluated. The abbreviation "ΔV2" as used herein refers to partial or complete deletion of the V2 domain. For a detailed description of the V2 domain of HIV-1, see Stamatatos, L .; , M .; Wiskerchen & C.I. Cheng-Mayer. 1998. Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV-1 isolate on viral environment structure, delivery-entry. AIDS Res. Hum. Retroviruses 14: 1129-1139, the description of which is incorporated herein by reference.
[0018]
Non-limiting means by which a modified protein comprising the HIV-1 envelope protein or a DNA encoding the modified protein can be prepared and deletions in the V2 region can be made are described in the above papers or in Stamatatos, L. et al. & C. Cheng-Mayer. 1998. An environmental modification that renders a primary, a neutralization resistant, and a clade B HIV-1 isolate high susceptible tolerance erosible erosion bye serothesis erosion bye J. Vifol. 72: 7840-7845. As a non-limiting example, a modified V2 deletion of the envelope protein of HIV-1 SF162 (clade B HIV-1) can be prepared, and the DNA and protein sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. However, other clade B HIV-1 envelope proteins can be similarly modified, and the protein or DNA encoding the protein can be used as an immunogen. Also, HIV-1 envelope proteins of other HIV-1 clades can be used. The selection of HIV-1 proteins and the amino acid sequences of their envelope proteins are described, for example, in http: // idiotype / lanl. It can be found in literature such as the Los Alamos National Laboratories HIV Sequence Database accessible at gov /. The present invention includes these and other HIV-1 envelope proteins as candidates for deletion in the V2 region for the preparation of a DNA or protein immunogen of interest herein.
[0019]
Standard methods of molecular biology can be used to prepare encoding DNA containing HIV-1 envelope proteins with deletions in the V2 domain, and viruses encoding the proteins. Is not limited with respect to the method of preparing the immunogen. The term DNA vaccine as used herein includes viral vaccines that contain DNA encoding the above proteins. Such methods are well-known in the art. As indicated herein, one of skill in the art can readily determine the ability of a DNA or protein immunogen of the invention to elicit a heterologous HIV-1 immune response in an animal. In a non-limiting example of the SF162 clade B HIV-1 virus strain, a 30 amino acid deletion of amino acids T160-Y189 is prepared and the deleted sequence is replaced with a Gly-Ala-Gly peptide. Substitution of the deleted sequence with the above-mentioned peptide or short peptide is not necessary but can be performed for convenience.
Animals capable of raising a heterologous viral immune response are those susceptible to an HIV-1 infection or a related virus. Such animals include, but are not limited to, humans, non-human primates, or other mammals. In the case of humans, the method of the invention can be performed on HIV-1, HIV-2, etc., and in non-human primates on SHIV-1.
[0020]
The present invention provides a vaccine pharmaceutical composition provided for immunizing an animal against the HIV-1 virus, comprising at least one modified HIV-1 envelope protein or a fragment thereof, wherein there is a V2 region deletion. A composition comprising DNA encoding one of the modified HIV-1 envelope proteins or fragments thereof, or a combination thereof, which is effective for raising a heterologous immune response, and a pharmaceutically acceptable carrier or excipient. About things. An immunogen, used interchangeably herein, may be a full-length or truncated modified protein or a DNA encoding a modified protein, provided that the V2 region elicits a heterologous immune response. Various choices for effective immunogens are described above. In embodiments, the modified HIV-1 envelope protein or fragment is from a Clade B HIV-1 strain. In a preferred embodiment, the HIV-1 strain is SF162. As a non-limiting example, the modified HIV-1 envelope protein or fragment is SEQ ID NO: 2 or SEQ ID NO: 4, and the DNA encoding at least one modified HIV-1 envelope protein or fragment is SEQ ID NO: 1 or SEQ ID NO: 3. It is. Glycosylation of proteins or fragments expressed in mammalian cells is also provided.
[0021]
A vaccine pharmaceutical composition can include one or more DNA or protein immunogens together with one or more pharmaceutically acceptable carriers, excipients, or diluents to facilitate administration of the vaccine. Additionally, additional components, such as one or more adjuvants, can be included to enhance the immune response. The choice of adjuvant will depend on the animal to be immunized, especially in humans for whom the choice of an appropriate adjuvant is limited. One skilled in the art can select appropriate pharmaceutically acceptable components to be included with the immunogen to achieve the desired effect.
It is a further object of the present invention to provide a method for assessing whether a compound, such as an immunogen, can produce protective antibodies against a heterologous strain of HIV-1. The method comprises immunizing an animal with an immunogen, depleting the animal's CD8 + T lymphocytes, and then presenting at least one protective antibody against at least one heterologous HIV-1 strain for the animal, preferably a protective antibody. By screening for the presence of The step of depleting may be performed by administering an anti-CD8 monoclonal antibody to the animal. The compound may be, but is not limited to, a HIV-1 derived polypeptide or a fragment thereof, for example, a DNA vaccine encoding an HIV derived polypeptide or a fragment thereof. Immunization protocols can include DNA vaccines, viral vaccines, proteins, fragments thereof, combinations thereof, or protocols in which one or both are administered sequentially to elicit an immune response. In a non-limiting embodiment, the neutralizing antibody is a protective antibody. Methods for assessing the induction of protective antibodies can be performed in animals capable of producing protective antibodies to HIV, such as, but not limited to, primates or other animals. As described above, the foregoing methods can also be used to evaluate the efficacy of the DNA and / or protein immunogens of the present invention.
[0022]
As described in the examples below, the lowest plasma peak viremia was recorded in animals vaccinated with the ΔV2 immunogen, and the serum had the strongest neutralizing activity against SHIV162P4 on the day of challenge. The finding that neutralizing antibodies played a significant role in protection during the first seven days after challenge. The fact that a strong anti-HIV envelope pre-response occurred immediately following SHIV162P4 challenge means that the antibody contributed to rapid virus clearance to undetectable levels. However, CD8 + lymphocytes reappeared in the periphery of the vaccinated animals 7 days post-challenge, which could also contribute to rapid virus clearance.
In addition, experiments of the present invention show an immune response to HIV-1 of a different clade than that from which the immunogen was prepared, referred to as a heterologous immune response.
These experiments highlight the important role of protection of non-CD8 mediated DNA-based vaccine-induced anti-HIV envelope responses and are viable to develop effective anti-HIV vaccines for human use in the prevention and treatment of HIV infection It proves the nature. As mentioned above, the strategy using a modified envelope protein with a ΔV2 loop deletion is a strategy that can be used for envelope proteins with a V2 loop, and the present invention is directed to all its uses, and for eliciting an immune response. A pharmaceutical composition comprising the ΔV2 loop deletion modified protein or DNA vaccine, or a combination thereof.
[0023]
In the experiments described herein, immunogenicity was compared between soluble oligomeric gp140 envelope proteins from related neutralization-resistant (SF162) and neutralization-sensitive (SF162ΔV2) viruses (Statatatos, L, & C. Cheng-Mayer, 1998, Envelope modification at end renders a primary, neutralization resist, clade B. HIV-1 ration s. The only difference between the two immunogens is the lack of 30 amino acids from the V2 loop of the SF162ΔV2-derived immunogen (Stamatatos, L, & C. Cheng-Mayer. 1998, An environmental modification renatura pratum. J. Virol. 72: 7840-7845) resistant, clade B HIV-1 isolated highly susceptible to neutralization by serum from other clades. Immunization experiments were first performed in rabbits, where both proteins elicited similar titers of bound antibody, whereas the modified immunogen was directed against isolates that expressed not only the SF162ΔV2 envelope, but also the unmodified parent SF162 envelope. It was found that a strong titer of neutralizing antibodies was elicited.
[0024]
In rabbits, both the unmodified SF162 gp140 immunogen and the modified ΔV2gp140 immunogen elicited neutralizing antibodies against some heterologous HIV-1 primary isolates, but the potential of such modified immunogens is even greater. Importantly, it was not previously described or expected. Thus, many animals vaccinated with the modified immunogen not only elicit cross-reactive neutralizing antibodies, but the breadth and intensity of the cross-neutralizing response is more collective from these animals than animals immunized with the unmodified immunogen. Was greater in serum. Modified immunogens elicit more effective antibody recognition neutralizing epitopes that are conserved among some HIV isolates than unmodified immunogens.
Vaccination experiments performed in rhesus monkeys confirm the findings made in rabbits, and the modified ΔV2gp140 immunogen is more effective than the unmodified SF162gp140 in raising neutralizing antibodies against isolates expressing the parent SF162 envelope. is there. Importantly, in monkeys, only the modified envelope is able to raise neutralizing antibodies against the heterologous HIV-1 isolate.
The present invention includes other envelope modifications in addition to the ΔV2 loop deletion described herein. Such modifications may increase the exposure and / or number of conserved neutralizing epitopes to the immunogen.
The following examples are provided to fully illustrate the preferred embodiment of the present invention. It should not, however, be construed as limiting the broad scope of the invention.
[0025]
Example 1
Two rhesus monkeys (Rh) (H445 and J408) were subjected to intact gp120-gp41 cleavage sites (Stamatatos, L., M. Lim & C. Cheng-Mayer. 2000. DNA vectors that express the SF162ΔV2gp140 envelope, which have the resistance and neutralization-susceptible primary HIV-1 isolates. AIDS Res. And Human Retroviruses. 16: 981-994, (Chap. . M. Thayer, K. A. Vincent & N. L. Haigwood 1991. Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologus expression in mammalian cells Nucleic Acids Res 19:.. 3979-86; zur Megede, J., MC Chen, B. Doe, M. Schaefer, CE Greer, M. Selby, GR Otten & SW W Barnett. 2000. Increasing technology-representation. modified hum . N immunodeficiency virus type 1 gag gene J Virol 74:. 2628-35) at week 0, (and all DNA) immunized with intradermal and intramuscular both of every 2 mg to 4 weeks and 8 weeks. Codons were optimized to enhance expression of the DNA construct in mammalian cells. At week 27, another immunization was performed with the DNA and CHO-producing purified oligomer SF162ΔV2gp140 protein (100 μg) mixed with MF-59C adjuvant. At week 38, another immunization was performed with the adjuvant mixed protein alone.
[0026]
The production of bound antibody was evaluated by ELISA (Stamatatos, L. & C. Cheng-Mayer. 1995. Structural modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding. J. Virol. 69: 6191-6198).
The antibody was detectable after the second DNA immunization and the titer did not increase after the third DNA immunization (FIG. 1). During the 5 months, the titer gradually decreased, but was always detectable. Approximate log from the peak value recorded after the third DNA immunization with the first 'boost'₁₀The titer increased by 1-2. Over the next 11 weeks, the titer gradually decreased and leveled off, at which point the animal was given a second 'boost' to further raise the antibody titer. Neutralizing antibodies (NA) were evaluated using an 'activated PBMC target' assay using pre-immune sera to correct for non-specific neutralization (Stamatatos, L. & C. Cheng-Mayer. 1998. An. Envelope modification that renders a primary, neutralization resistant, and clade B HIV-1 isolate high sucdpptible 78 thr. After the third DNA immunization, the bound antibody titers were the same between the two animals, but the H445 animals had lower NA titers than the J408 animals. Following the 'boost', both NA titers to SF162ΔV2 and SF162 increased significantly. Vaccine-specific proliferative responses were recorded in all animals. J408 and H445 animals recorded stimulation indices (SI) of 5 and 10 after the first 'boost', respectively. The second 'boost' increased the response strength of H445 animals (SI.25), but not J408 animals (SI.5).
[0027]
To assess the protective role of high HIV envelope antibodies elicited by the vaccine of the present invention, CD8 + cells were depleted from vaccinated animals prior to virus challenge (FIG. 3). Intravenous administration of anti-CD8 MAb OKT8F (2 mg / kg) three times every other day resulted in CD8 depletion (Jin, XDE Bauer, SE Tuttleton, S. Lewin, A. Gettie, J. Blanchard, CE Irwin, JT Safrit, J. Mittler, L. Weinberger, L. G. Kostrikis, L. Z. D. S. H., A. Z. D. S. D.S. 1999. Dramatic rise in plasma viremia after CD8 (+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp9 Ed Exp9. -8). CD8 + T lymphocytes remained undetectable in about 10 days. Concomitantly, a decrease in the total number of circulating CD3 + T cells was recorded. This means that the recording of CD8 + T cell depletion from the periphery is due to actual detachment. Although CD8 depletion from lymph nodes was not evaluated, it has previously been demonstrated that the introduction of anti-CD8 MAbs into the blood circulation of monkeys results in a concomitant depletion of CD8 + T cells from peripheral and lymph nodes (Matano). , T., R. Shibata, C. Siemon, M. Connors, H. C. Lane & M. A. Martin. 1998. Administration of an anti-CD8 monoclonal antibody interfers with the clearance of chimeric simian / human immunodeficiency virus during J. Virol. 72: 164: 16, primary infections of rhesus macaques. Schmittz, JE, MJ Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, Mal. BJ J. Scallon, J. Ghrayeb, M. A. Forman, D. C. Montefiori, E. P. Rieber, N. L. Letvin & K. A. Rei. Rem. by CD8 + lymphocytes. Science. 283: 857-60).
[0028]
One day after the last dose of OKT8F, immunized or two non-immunized naive animals were treated with 100 TCID₅₀Intracellularly challenged with a cell-free strain of the SHIV162P4 virus (Harouse, JM, A. Gettie, RC Tan. J. Blanchard & C. Cheng-Mayer. with CCR5 or CXCR4 utilizing SHIVs. Science. 284: 816-9). This isolate was 50% and 90% neutralized by serum (diluted 1: 5) collected on the day of challenge from H445 and J408 animals, respectively.
Both vaccinated and non-vaccinated animals became infected. However, there was a difference between the peak virus load level and the virus set point between the two groups (FIG. 4A). At 11 days post-challenge, plasma viremia in vaccinated H445 animals was log compared to unvaccinated A141 and AT54 animals, respectively.₁₀J408 animals vaccinated were 2 and 4 low and were aviremia. At the viremia peak, virus plasma levels in vaccinated animals are logarithmically greater than in unvaccinated animals.₁₀It was 1-4 lower. After the viremia peak, a gradual decline in plasma viral load was recorded in the unvaccinated A141 animals following the first steep decline, while a second unvaccinated AT54 animal had a high viral load. Persistence has been recorded. Very rapid declines to undetectable levels were recorded in both animals vaccinated within 35 days of challenge.
[0029]
Concomitant with the appearance of plasma viremia in vaccinated H445 animals, a rapid increase in anti-HIV envelope antibody titers (only about 5-fold) was monitored (FIG. 4B). The antibody titer then decreased gradually to the pre-challenge titer, as did the animal's viral load to undetectable levels. In contrast, the second vaccinated J408 animals did not increase anti-envelope antibody titers and had the lowest plasma viremia peak. In non-vaccinated animals, anti-HIV envelope antibodies became detectable approximately 30 days after challenge. Titers increased over time in A141 animals, but diminished in AT54 animals, which remained weak and eventually declined.
Two unvaccinated animals seroconverted to SIV gap p27 and pol 31 proteins within two weeks after challenge, and two vaccinated animals remained seronegative for the first 17 weeks after challenge (FIG. 5). This figure shows the conversion of the core SIV proteins to the gap 27 and pol 31 and gp41 and gp120 HIV envelope subunits, as determined by RIBATM. The numbers in each strip above indicate the day the serum samples were collected relative to the day of challenge (day 0) [(+) positive control strip; (-) negative control strip].
[0030]
Also, virus was recoverable from Rh-PBMC collected from unvaccinated animals on days 18, 42 and 48 post-challenge, but only from day 18 on vaccinated animals. there were. Finally, in contrast to the two vaccinated and non-vaccinated animals A141, which remained healthy, the second non-vaccinated AT54 animal died from SAIDS 16 weeks after challenge.
The finding that the lowest level of plasma viremia peak was recorded in vaccinated J408 animals whose sera had the strongest neutralizing activity against SHIV162P4 on the day of challenge, suggests that neutralizing antibody Played an important defense role. However, in addition to the neutralizing antibody, an envelope-specific antibody containing no neutralizing activity can be raised by the vaccine of the present invention, and can contribute to virus clearance. The fact that a strong anti-HIV envelope pre-existing response occurs in H445 animals immediately after SHIV challenge means that the antibody contributed to rapid virus clearance to undetectable levels. However, as CD8 + lymphocytes reappeared in the periphery of vaccinated animals 7 days after challenge, they can also contribute to their rapid viral clearance.
These experiments highlight the important protective role of non-CD8 mediated DNA vaccine-induced anti-HIV envelope responses and demonstrate the feasibility of developing effective anti-HIV vaccines.
[0031]
Example 2
In the experiments shown here, the potential immunogenicity of unmodified SF162 is compared to a modified SF162ΔV2 (hereinafter ΔV2) envelope. Rabbits were immunized with SF162 and the gp140 form of the ΔV2 envelope using the gene gun vaccination method. Both immunogens elicited generation of similar antibody titers, whereas the modified immunogen elicited higher titers of neutralizing antibodies against the parent SF162 virus than the unmodified immunogen. In addition, the ΔV2-derived modified immunogen was more effective than the SF162-derived unmodified immunogen in producing antibodies capable of neutralizing heterologous HIV-1 primary isolates.
The immunogenicity of these two antigens was assessed using DNA priming followed by a protein boost vaccination method in rhesus monkeys in animal models closely related to humans and suitable for HIV vaccine experiments. In this regard, the modified immunogen was found to be more effective than the unmodified immunogen in producing potent neutralizing antibodies against both the homologous SF162ΔV2 virus and the parent SF162 virus. Antibodies were elicited in the monkeys with the modified immunogen and those that were not elicited with the unmodified immunogen neutralized some heterologous HIV-1 primary isolates. These experiments show for the first time that potent cross-reactive neutralizing antibodies can be elicited in non-human primates immunized with a soluble oligomeric subunit HIV envelope vaccine derived from an HIV-1-like primary isolate using R5. Is shown. It supports the use of specific envelope modifications to increase the exposure of neutralizing epitopes and to increase the breadth and strength of these responses.
[0032]
Viruses: Separation and phenotypic confirmation of SF162 and SF162V2 has been previously reported (Cheng-Mayer, C., M. Quiroga, JW Tung, D. Dina & J.A. Levy 1990. Viral. determinants of human immunodeficiency virus type 1 T-cell or macrophage tropism, cytopathogenicity, and CD4 antigen modulation, J. Virol 64:.. 4390-4398; Stamatatos, L. & C. Cheng-Mayer 1998. An envelope modification that renders a primary, neutrali . Ation resistant, clade B HIV-1 isolate highly susceptible to neutralization by sera from other clades J. Virol 72:. 7840-7845). Clade B HIV-1 primary isolates 92US660, 92HT593, 92US657, 92US714, 92US727, 91US056, 91US054 and 93US073 were obtained from the NIH AIDS Research and Reference Reagent program. All virus strains were prepared and titered in activated human peripheral blood mononuclear cells (PBMC).
[0033]
Vaccine: The DNA vector used to express the immunogens of the present invention in rabbits is pJW4303 (Lu, S., Wyatt, JFL Richmond, F. Mustafa, S. Wang, J. Weng, D. C. Montefiori, J. Sodroski & H. L. Robinson. 1998. Immunogenicity of DNA vaccines expressing human immunodeficiency virus type 1 envelope glycoprotein with and without deletions in the V1 / V2 and V3 regions. AIDS Res. Hum. Retroviruses 14 : 151-155)The DNA vector used to immunize rhesus monkeys is derived from the pCMVKm2 vector (Chapman, BS, RM Thayer, K. A. Vincent & N.L. Haigwood. 1991. Effect of introduction. Cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammarian cells. Nucleic Acids Res., 19: 3979-86, zur Med. , M. Selby, GR Otten & SW Barnett. 2000.I. J. Virol. 74: 2628-35. Both plasmids have a human CMV enhancer / promoter element and the native leader of the HIV envelope was replaced with that from the tissue-specific plasminogen activator gene. For monkey immunization, codons were optimized to enhance expression of the DNA construct in mammals. Both DNA vectors express the gp140 ectodomain of the HIV envelope immunogen with an intact gp120-gp41 cleavage site.
[0034]
Protein boosts were performed only in rhesus monkeys to enhance the immunization of the DNA phase following the titer of the eliciting antibodies. For this, the ΔV2gp140 protein was produced in CHO cells and purified as a stable soluble trimer. However, to increase the stability of these secreted oligomers, the gp120-gp41 cleavage site was removed by mutagenesis (Earl, PL, S. Koenig & B. Moss. 1991. Biological and immunological properties of humans). . immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses J Virol 65:. 31-41; Earl, P. L. & B. Moss 1993. Mutational analysis ... F the assembly domain of the HIV-1 envelope glycoprotein AIDS Res Hum Retroviruses 9:. 589-594; Stamatatos, L., M. Lim & C. Cheng-Mayer 2000. Generation and structural analysis of soluble oligomeric envelope proteins Derived from neutralization-resistant and neutralization-susceptible primary HIV-1 isolates. AIDS Res. Hum. Retroviruses 16: 981-994).
[0035]
Immunization: a) Rabbit: Gene cancer vaccination method (Lu, S., R. Wyatt, J. F. L. Richmond, F. Mustafa, S. Wang, J. Weng, D. C. Montefiori, J. Mol. Sodroski & H. L. Robinson 1998. Immunogenicity of DNA vaccines expressing human immunodeficiency virus type 1 envelope glycoprotein with and without deletions in the V1 / V2 and V3 regions AIDS Res Hum Retroviruses 14:.... 151-155) by using the Animals received 5 weeks at weeks 0, 4, 8, 18, and 22. DNA immunization was performed once. Blood was drawn two weeks after each immunization. Six animals (A1-A6) were immunized with the modified SF162 gp140 immunogen, and six animals (A7-A12) were immunized with the modified ΔV2gp140 immunogen. Two animals (A13 and A14) served as controls and were immunized with DNA vector only.
[0036]
b) Rhesus monkeys: H445 and J408 animals were immunized with the modified ΔV2gp140 immunogen, N472 and P655 animals were immunized with the unmodified SF162 gp140 immunogen, and M844 and H473 animals were immunized with the DNA vector only. Prior to the start of immunization, animals were tested for antibodies against various simian viruses such as SIV, D-type retrovirus or STLV-1. Animals vaccinated with the modified envelope were immunized with DNA at weeks 0, 4, and 8, and animals vaccinated with the unmodified envelope were immunized with DNA at weeks 0, 4, and 9. DNA (2 mg of DNA in 1 ml of endotoxin-free water per animal each time) was injected intradermally (id) at two sites (2 × 0.2 mg) and intramuscularly (im) (iv). The head muscle was administered both at 2 × 0.8 mg). A fourth time the animals were immunized with DNA and simultaneously with purified oligomer ΔV2gp140 or SF162gp140 protein mixed with MF-59C adjuvant. Protein (0.1 mg of purified protein in a total volume of 0.5 ml per animal) was injected i.p. m. Administration. Control animals received only adjuvant. This DNA + protein 'booster' immunization occurred in animals vaccinated with the modified immunogen at 27 weeks and in animals immunized with the unmodified immunogen at 48 weeks. Animals immunized with the modified immunogen at week 38 were immunized once more with only the adjuvant mixed protein (no DNA), and none of the animals immunized with the unmodified immunogen.
[0037]
Antibody quantification: a) Anti-gp140 antibody: The titer of the entire immunization protocol was quantified using the ELISA method as previously described (Stamatatos, L. & C. Cheng-Mayer. 1995. Structural modulations of the). .. envelope gp120 glycoprotein od human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding J. Virol 69: 6191-6198; Stamatatos, L., M. Wiskerchen & C. Cheng-Mayer. 1998. Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV-1 isolate on viral envelope structure, cell-entry and replication. AIDS Res. Hum.Retroviruses 14: 1129-1139). Briefly, ELISA plates (Immulon 2HB) (0.2 μg protein in 0.1 ml 100 mM NaHCO3, pH 8.5) were incubated overnight at 4 ° C. with purified soluble oligomer ΔV2gp140 and SF162gp140 proteins. By coating. Unadsorbed protein molecules were removed with TBS and wells were blocked with Superblock (SB) (Pierce). Heat inactivation (35 min at 56 ° C.) collected from immunized animals was serially diluted in SB and added to wells for 1 h at 37 ° C. (0.1 ml / well). In the case of rabbits, sera from control animals that received only the DNA vector were used as negative controls.
[0038]
In monkeys, pre-immune serum was used as a negative control. Unbound antibody was removed by TBS washing, and the envelope-bound antibody was isolated from goat anti-human (for monkey serum) or anti-rabbit (rabbit serum) conjugated to alkaline phosphatase antibody (Zyme Immunochemicals) as previously described. of the case) it was detected by using a (Stamatatos, L. & C. Cheng-Mayer. 1995. Structural modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct trop J. Virol. 69: 6191-6198), ismupon virion-soluble receptor binding. The OD 490 nm of each well was recorded with a bioluminometer (Molecular Dynamics). A plot of OD 490 nm against serum dilutions was made and endpoint antibody titers were determined as the highest post-immune serum dilution yielding an OD 490 nm of 3 times the OD 490 nm generated with the lowest dilution pre-immune serum. At the same time, sera from each stage of immunization were tested.
[0039]
Neutralization Assay: Neutralization analysis was performed as previously described using human PBMCs activated with PHA (Sigma, 3 μg / ml) for 3 days as target cells (Mascola, JR, M.). G. Lewis, G. Stiegler, D. Harris, TC Vancott, D. Hayes, M.K. Louder, CR. Brown, C.V. Sapan, S.F.San Francisco. , M.L. Robb, H. Katinger & D.L.Birx.1999. Protection of Macaques against pathogenic simian / human immunodefiency virousairvous. J. Virol. 73: 4009-18; Mascola, JR, SWW Snyder, OS Weislow, SM Belay, RB Belsh, D.H. ML Clements, R. Dolin, B.S. Graham, G.J.Gorse, M.C. Keefer, M.J. McElrath, M.C. Walker, K.F. Wagner, J.G. McNeil, F.E. McCutchan, DS Burke & the NIAID AIDS vaccine evaluation group. 1996. Immunization with environmental investment. .. Ts elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. J. Infect Dis 173:. 340-348; Stamatatos, L. & C. Cheng-Mater 1998. An envelope modification that renders a primary, neutralization resistant, and grade B HIV-1 isolated high susceptible to neutralization by serum from other grades. J. Virol. 72: 7840-7845; Stamatatos, L .; , S.M. Zolla-Pazner, M.A. Gorny & C.E. Cheng-Mayer. 1997. Binding of antibodies to virion-associated gp120 molecules of primary-like human immunodeficiency virus type 1 (HIV-1) isolates: effect on HIV-1 infection of macrophages and peripheral blood mononuclear cells. Virology 229: 360-369).
[0040]
All of the HIV-1 isolates to be tested were grown, titrated in human PBMC, aliquoted and frozen at -80 ° C until use. Virus (50-100 TCID in 50 μl RPMI complete medium containing 20 U / ml IL-2₅₀(Hoffman Laroche)) was preincubated with the same volume of serially diluted heat-inactivated (56 ° C. for 35 minutes) serum for 1 hour at 37 ° C. in a 96-well U-bottom plate (Corning). For each serum dilution, triplicate wells were used. Pre-immune serum from monkeys and serum collected from rabbits immunized with DNA vector only were incubated with virus and served as non-specific neutralization controls. 0.4 × 10 per well⁶0.1 ml of complete medium containing PHA-activated PBMC was added. After overnight incubation at 37 ° C., half of each well was replaced with fresh RPMI complete medium. After centrifuging the plate (2,000 rpm for 5 minutes), half of each well was replaced with fresh medium again. This procedure was repeated twice. The p24 antigen concentration in each well was evaluated at various time points post infection (usually on days 4, 6, and 11) using an in-house ELISA p24 assay analysis. The mean% neutralization and the standard deviation of each serum dilution from the wells of triplicate were previously described based on the p24 concentration recorded in the wells containing virus, cells, and non-rabbit or non-monkey serum. (Stamatatos, L.S. Zolla-Pazner, M. Gorny & C. Cheng-Mayer. 1997. Binding of antibodies to voyage-economy gasoline physics and physics- -1) isolates: effect on HIV-1 effect of macrophages and peripheral blood mononu . Lear cells Virology 229: 360-369).
[0041]
However, it was observed that infection of some isolates was reduced in the presence of pre-immune serum (non-specific neutralization). Therefore, results are shown from two methods of neutralization experiments. 1. The same drawing shows both the neutralization curves recorded with sera collected before vaccination (prior to bleeding) and with sera collected at various stages after vaccination. I have. 2. For each serum dilution, the difference between the% neutralization recorded in the serum after vaccination-the% neutralization recorded in the serum before vaccination was calculated. In some figures, this difference (referred to herein as "specific neutralization") is plotted as a function of serum dilution. At the same time, the sensitivity of various primary isolates to neutralization by MAbs 2F5 and 2G12 was evaluated.
[0042]
In these neutralization experiments, the performance of sera collected from monkeys immunized with the recombinant SF2 gp120 envelope was evaluated. This immunogen was previously tested as a potential vaccine against HIV and failed to raise cross-reactive neutralizing antibodies (Mascola, JR, SWW Snyder, OS Weilow). , SM Belay, RB Belshe, DH Schwartz, ML Clements, R. Dolin, B.S. Graham, G.J.Gorse, M.C.Keefer, M.K. McElrath, MC Walker, K. F. Wagner, J. G. McNeil, F.E. McCutchan, DS Burke & the NIAID AIDS vaporization evolution 96. .. E subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. J. Infect Dis 173: 340-348).
[0043]
Results: Production of Antibodies in Rabbits: Both SF162-derived and ΔV2 immunogens elicited high titers of antibodies that could bind to both oligomeric ΔV2gp140 and SF162gp140 proteins (FIG. 6). As expected, changes in antibody titers were recorded throughout the vaccination regime of animals belonging to either group. However, no statistically significant antibody titer differences were recorded between the two groups of animals throughout the immunization program. The antibody titers in each animal, whether immunized with the modified or unmodified immunogen, were very weak during the first two immunizations (week 0 and week 4). The fourth immunization (week 18) resulted in a log increase in both animal groups compared to the third immunization (week 8).₁₀An increase in antibody titer was obtained with a few. The fifth immunization (week 22) compared to the fourth immunization with the SF162 gp140 antigen (log₁₀Antibody titer (only less than 1), but not the ΔV2gp140 protein. At the end of the vaccination program, both groups of animals received 10⁵-10⁶A very strong endpoint ELISA binding antibody was recorded with a degree of. Thus, in rabbits, based on the analysis used here to determine antibody titers, the modified immunogen lacks 30 amino acids from the V2 loop but is unmodified immunogenic to elicit antibody production. It seems to be as effective as Hara.
[0044]
Neutralizing antibodies in rabbit sera against SF162 isolate and SF162ΔV2 isolate: Both immunogens produced neutralizing antibodies against SF162ΔV2 virus after the third immunization (FIG. 7A). A trend for higher neutralizing titers was recorded in the modified immunogen vaccination group. Therefore, the mean serum dilution (and standard error) recorded for animals immunized with SF162gp140 and SF162ΔV2 with 70% inhibition were 179 (+/− 34) and 483 (+/− 148), respectively. At the time of this vaccination, 2 out of 6 (A8 and A9) immunized with the modified immunogen elicited neutralizing antibodies against the parental SF162 isolate, whereas the animals immunized with the unmodified immunogen did not None raised antibodies that could do so (FIG. 7B). However, the number of animals that produced neutralizing antibodies to the SF162 and SF162ΔV2 viruses each increased with subsequent immunizations, so that at the end of the immunization plan (ie, after the fifth immunization), all animals were SF162 Neutralizing antibodies were produced against the virus. Furthermore, the neutralizing potency of each serum was increased with each immunization, regardless of whether the animals were vaccinated with the modified or unmodified immunogen.
[0045]
At the end of the immunization scheme, sera collected from rabbits immunized with the modified immunogen were more neutralizing than antisera collected from animals immunized with the unmodified immunogen against SF162ΔV2 and SF162 viruses. Six of the six animals immunized with the modified immunogen elicited 70% to 100% of antibodies capable of neutralizing the SF162ΔV2 virus at a 1: 5,000 dilution (FIG. 7A). In contrast, only one of the six animals (A1) vaccinated with the unmodified envelope at the same serum dilution produced an antibody response that was able to neutralize SF162ΔV2 infection by 50%. The remaining five animals in this group failed to elicit an antibody response that sufficiently neutralized SF162ΔV2 infection with this dilution to a significant degree. The difference in neutralization potential between sera collected from animals immunized with the modified immunogen and those immunized with the unmodified immunization was evident when comparing their ability to neutralize the SF162 virus (FIG. 7B). ). Sera collected from 4 of 6 animals (A8, A9, A10 and A12) immunized with the modified antigen neutralized 70% to 90% SF162 infection with 1: 100 to 1: 300 dilutions did. In contrast, none of the sera collected from animals immunized with the unmodified antigen was able to block SF162 infection by only 70% -90% at the same dilution.
[0046]
Production of Cross-Reactive Neutralizing Antibodies in Rabbits: Envelope immunogen from SF162ΔV2 has higher titer of neutralizing antibody against parent SF162 isolate (expressing the full envelope) than immunogen from SF162 isolate itself The fact that it was able to elicit was to test whether the modified immunogen was effective in raising cross-reactive neutralizing antibodies, ie, antibodies that could neutralize a heterologous HIV-1 primary isolate against the vaccine. Ushered us. Several such isolates with previously demonstrated neutralization sensitivity to various monoclonal antibodies were tested (D'Souza, MP, D. Livnat, JA Bradac & SH). Bridges 1997, Evaluation of monoclonal antibodies to human immunodeficiency virus type 1 primary isolates by neutralization assays:.. performance criteria for selecting candidate antibodies for clinical trials AIDS Clinical Trials Group Antibody Selection Working Group J. Infect. Dis. 175: 1056-62). Only two of the six isolates (92US714 and 92HT593) were neutralized with antibodies raised by the unmodified immunogen (Table 1 below).
[0047]
[Table 1]

[0048]
Neutralizing activity was evaluated at a 1:10 dilution and took into account non-specific neutralization recorded in sera collected from animals vaccinated with the DNA vector alone (see Materials and Methods for details). ). (-): 50% specific neutralization was not recorded. (+): 50% to 80% specific neutralization was recorded. Results are from three independent neutralization experiments.
With the exception of the A1 animal, all other animals produced neutralizing antibodies against 92US714, and only the A2 and A5 animals produced neutralizing antibodies against 92HT593. In contrast, four out of six animals immunized with the modified ΔV2gp120 immunogen produced cross-reactive neutralizing antibodies against most of the heterologous isolates tested. In addition, the intensity of neutralization of sera collected from animals immunized with the modified immunogen was greater than sera collected from animals immunized with the unmodified immunogen (see Table 1 above). Thus, while 80% inhibition of infection was often recorded with the former sera, this level of inhibition was recorded only in two cases (serum from A5 animals against isolates of 92US714 and 92HT593).
[0049]
Antibody production in rhesus monkeys vaccinated with the modified ΔV2gp140 immunogen: The above results indicate that the immunogenicity of the unmodified SF162gp140 antigen and the modified ΔV2gp140 antigen in rhesus monkeys in an animal model that could ultimately assess the protective potential of the vaccine-evoked antibodies Driven gender evaluation. Monkeys were vaccinated with these two immunogens using a DNA boost followed by a protein boost.
Envelope specific antibodies were detectable after the second DNA immunization (FIG. 8). At this stage, the endpoint ELISA titers in animals immunized with the modified antigen (J408 and H445 animals) were on the order of 1: 2,000. In contrast, in animals immunized with the unmodified envelope (N472 and P655 animals), antibodies were only detectable in animal N472 (end point ELISA titer on the order of 1: 500). With the exception of H445 animals, the third DNA immunization did not increase antibody titers. Anti-gp120 and anti-gp41 antibodies were produced synchronously by DNA immunization.
Subsequent 5-10 month observations show that no antibody is detectable in animals immunized with the unmodified SF162 gp140 immunogen, and in animals immunized with the modified ΔV2gp140 immunogen, the antibody is always detectable, but its titer is Decreased over time.
[0050]
Following DNA + protein 'boost' immunization, antibody titers increased significantly in all animals. At peak values (reached within 2-4 weeks after 'boost'), endpoint ELISA antibody titers in animals immunized with the modified ΔV2gp140 immunogen were 1: 30,000 for J408 animals and for H445 animals. 1: 110,000. The titer gradually decreased over time, and both animals were stable at about 1: 8,000 for several weeks. High peak antibody titers were recorded in animals vaccinated with the unmodified SF162 gp immunogen (end point ELISA antibody titers 1: 150,000 for animals with N472 and 175,000 for animals with P655). In the next 7 weeks, both animals showed a rapid drop in antibody titer to about 1: 35,000. Thus, in monkeys, in contrast to those recorded in rabbits, the unmodified immunogen produced higher titers of bound antibody than the modified immunogen.
As expected, no anti-HIV envelope antibody was produced in control animals (M844 and H473) immunized with the DNA vector alone.
Neutralizing activity of monkey serum against allogeneic SF162ΔV2 isolate and parental SF162 isolate: In the DNA phase immunization, only animals immunized with the modified ΔV2gp140 elicited neutralizing antibodies against SF162 and SF162ΔV2 viruses (FIG. 9A- B). After the second DNA immunization, the J408 animals produced neutralizing antibodies against the allogeneic SF162ΔV2 isolate but not the parental SF162 isolate (FIG. 9A). The titer of neutralizing antibody in J408 animals increased after the third DNA immunization, at which point neutralization of both isolates was recorded, but the titer of bound antibody did not increase simultaneously (FIG. 9B). ). In contrast, a very weak neutralizing antibody response was elicited against the SF162ΔV2 virus in H445 animals and no neutralizing antibody response was elicited against the SF162 virus, but the animals produced J408. A binding antibody with the same titer as was produced (FIG. 9B).
[0051]
Two weeks after DNA + protein 'booster' immunization, sera collected from animals immunized with either immunogen blocked SF162ΔV2 infection. The neutralization strength of sera collected from animals immunized with the modified immunogen was greater than sera collected from animals immunized with the unmodified immunogen. For example, 50% inhibition of SF162ΔV2 infection was recorded at a dilution of 1: 2,000 to 1: 5,000 from the former serum, but this level of inhibition was observed in serum collected from the latter serum at this dilution. Was not recorded in. Although all ΔV2gp140 immunized animals produced strong neutralizing antibodies against the parental SF162 virus, only one of the two animals (N472) immunized with the SF162 gp140 immunogen produced neutralizing antibodies against this virus. did. No changes in the neutralization intensity of these sera were recorded in the next two weeks, but changes in antibody titer levels were detectable during this period (FIG. 9). Control animals vaccinated with the vector alone (M844 and H473) did not produce neutralizing antibodies.
Neutralization of heterologous HIV-1 primary isolates by monkey serum: The range of neutralizing antibody responses elicited in monkeys immunized with the modified and unmodified immunogens indicates that infection of the heterologous Clade B HIV-1 primary isolates The ability of sera collected from monkeys immunized with these two immunogens to block was assessed by comparing. The sensitivity of these isolates to be neutralized by various MAbs has been reported previously (D'Souza, MP, D. Livnat, JA Bradac & SH Bridges. 1997. Evaluation of monoclonal antibodies to human immunodeficiency virus type 1 primary isolates by neutralization assays:.. performance criteria for selecting candidate antibodies for clinical trials AIDS Clinical Trials Group Antibody Selection Working Group J. Inf ect. Dis. 175: 1056-62). Serum neutralization experiments evaluated the sensitivity of these isolates to be neutralized simultaneously by the two most commonly used primary isolate neutralizing MAbs (2F5 and 2G12) (Table 2).
[0052]
[Table 2]

[0053]
Values are constant HIV-1 with serum (1:10 dilution) collected from animals immunized with modified ΔV2gp140 (J408 and H445), unmodified SF162 gp140 (P655 and N472) and recombinant gp120 (L714 and L814). % Neutralization of the isolate. The use of the co-receptor for each isolate is indicated in parentheses. Percent neutralization was calculated as described in Materials and Methods and took into account non-specific neutralization recorded in sera collected from the same animals prior to the start of the immunization protocol.^&(A): DNA collected from J408 and H445 animals + sera 2 weeks after protein 'boost' immunization and (B) serum collected 2 weeks after last protein 'booster' immunization. Values are averages from 2-3 independent experiments. The susceptibility of these isolates to neutralization of 2F5 and 2G12 with a 25 μg / ml MAb is also shown. NT: Not evaluated.
Immunization of the DNA phase in monkeys did not record heterologous isolate neutralization (less than 50% inhibition at 1:10 dilution). Two weeks after DNA + protein 'booster' immunization, sera collected from two animals vaccinated with the modified ΔV2gp140 protein neutralized some of the heterologous HIV-1 primary isolates tested (FIG. 10). At the lowest serum dilution tested (1:10) and taking into account the non-specific neutralization recorded in the pre-immune sera (see Materials and Methods for details), 80-90% Inhibition was recorded only for the ADA, 91US056 and 92US714 isolates by J408, and only for the ADA, 92US714 and 92US660 isolates by H445 serum (FIG. 10 and Table 2). Cross-neutralizing activities of sera collected from these two animals differed. For example, 92US660 infection was blocked by sera of H445 and J408 by 80% and 50%, respectively. Serum cross-neutralizing activity was reduced on subsequent weekly findings (FIG. 10). Sera collected 5 weeks after this DNA + protein 'booster' immunization had no cross-reactive neutralizing activity, but strong neutralization of the isolates of SF162 and SF162ΔV2 was still recorded.
[0054]
Following this DNA + protein 'boost' immunization, despite the fact that the bound antibody titers in animals vaccinated with the unmodified immunogen were greater than those vaccinated with the modified immunogen (Figure 8), the former serum was tested. None of the heterologous isolates could be neutralized (Table 2) (ie, less than 50% specific neutralization was recorded). Thus, in rabbits, the unmodified immunogen was able to elicit neutralizing antibodies against some heterologous HIV-1 primary isolates (albeit much less efficient than the modified immunogen) (Table 1). In rhesus monkeys it could not be so induced.
At the same time, the sensitivity of the heterologous isolate neutralized by serum collected from monkeys immunized with the recombinant SF2-derived gp120 protein was evaluated. This protein was previously evaluated as a vaccine candidate and was ineffective at raising cross-reactive neutralizing antibodies. That is, less than 50% neutralization was recorded with a 1:10 serum dilution (Mascola, JR, SW Snyder, OS Weislow, SM Belay, RB). Belshe, DH Schwartz, ML Clements, R. Dolin, BS Graham, GJ Gorse, MC Keefer, MJ McElrath, M.K.Walk. F. Wagner, J. G. McNeil, F.E. McCutchan, DS Burke & the NIAID AIDS vaccine evaluation group. 1996. Immunization investment payments. It's neutralizing antibodies against laboratory-adapted but not primary isolations of human immunodeficiency, virus type 1. J.3. All isolates tested here were not sensitive to neutralization by antibodies elicited by the SF2 gp120 protein (Table 2).
[0055]
Second 'boost' immunization with the modified ΔV2gp140 protein: The above results showed that the modified ΔV2gp140 immunogen was actually more effective at eliciting a cross-reactive neutralizing antibody response than the unmodified immunogen. Response was weaker than that recorded for the parental SF162 isolate (FIGS. 11A-B). In an effort to further increase the strength and breadth of these responses, an attempt was made to further 'boost' antibody titers in H445 and J408 animals by a single additional immunization with the purified oligomer ΔV2gp140 protein. There is no DNA immunity).
Since the increase in antibody titer was actually recorded after this protein 'boost', the peak (1: 145,000 endpoint ELISA titer) titer was recorded during the first 'booster' immunization with DNA + protein. Approximately three times as large as that of Fig. 11A. At the same time, there was a marked increase in the titer of neutralizing antibodies against the homologous SF162ΔV2 isolate and the parent SF162 isolate (FIG. 11).
No difference in neutralization potential of sera collected two and five weeks after this last 'boost' was reported, but bound antibody titers were significantly reduced during the same period. However, unexpectedly, the neutralization potential of the same serum against most of the heterologous primary isolates tested was often reduced (Table 2). Thus, except for the isolates of BZ167, 92US657 and ADA, all of the heterologous isolates tested resisted neutralization by serum collected two weeks after the second 'boost'. Interestingly, isolate 92US657 resisted neutralization with serum collected after the first boost, but was susceptible to neutralization with serum collected after the second boost.
[0056]
Production of anti-V3 loop antibodies in rhesus monkeys vaccinated with modified ΔV2gp140: explanation for increased neutralizing activity against parental SF162 virus and allogeneic SF162ΔV2 virus and decreased neutralizing activity against heterologous isolates after the second 'boost' immunization Multiple immunization with the modified ΔV2gp140 protein increases the titer of antibodies to the SF162 envelope and to an epitope that is uniquely (or preferentially) expressed on the SF162ΔV2 envelope. It is likely that multiple immunizations with ΔV2gp140 will result in the production of high titers of anti-V3 loop antibody. To determine the titer of such antibodies, a V3 loop peptide-based ELISA analysis using a SF3 / SF162ΔV2-derived V3 loop was used (FIGS. 12A-B). This peptide has a linear (447D) epitope (Conley, AJ, MKK Gorney, JA Kessler, second, LJ Boots, M. Ossorio-Castro, S. Koenig, D.). . W. Lineberger, E. A. Emini, C. Williams & S. Zolla-Pazner. 1994. Neutralization of primary human immunodeficiency virus type 1 isolates by the broadly reactive anti-V3 monoclonal antibody, 447-52D. J. Virol. Gorny, MK, AJ Conley, S. 68: 6994-7000; . Karwowska, A. Buchbinder, J.-Y. Xu, E. A. Emini, S. Koenig & S. Zolla-Pazner. 1992. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. J. Virol. 66: 7538-7542) and also a conformational (391-95D) epitope (Seligman, SJ, JM Binley, MKK Gorny, DR Burton, S. Zolla). -Pazner & K. A. Sokolowski 1996. Characterization by ... Erial competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies Mol Immunol 33: 737-745 also) recognized by antibodies that bind (Figure 11A).
[0057]
Anti-V3 loop antibodies were produced upon immunization of monkeys with the modified ΔV2gp140 immunogen, but the titers were much lower than for the whole envelope (FIG. 11B). Furthermore, the second 'boost' immunization did not increase the titer of the anti-V3 loop antibody. However, although certain anti-V3 loop antibodies present in the sera of these animals are unable to interact efficiently with V3 loop peptides in an ELISA format, they cannot bind to epitopes on the native envelope. it should be noted (Moore, J. P. 1993. the reactivities of HIV-1 + human sera with solid-phase V3 loop peptides can be poor predictors of their reactivities with V3 loops on native gp 120 molecules. AIDS Res. Hum. Retroviruses 9: 209-19). Furthermore, the V3 loop peptide used here does not span the carboxy and amino termini of the V3 loop, and analysis does not detect antibodies targeting these two regions. Therefore, it is necessary to examine in more detail the epitope specificity of antibodies raised with the modified ΔV2gp140.
[0058]
Figures 13A and 13B are graphs showing the heterogeneous immune response elicited by the immunogens of the present invention by neutralizing different clade HIV-1 viruses. FIG. 13A shows neutralization using serum from H445 animals, and FIG. 13B shows neutralization using serum from J408.
Although the invention has been described and illustrated by way of individual embodiments, individual materials, procedures and examples, it should be understood that the invention is not limited to the specific material combinations of materials or procedures selected therefor. Is understood. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various documents are cited herein, the disclosures of which are incorporated herein.
[Brief description of the drawings]
FIG.
Figure 4 is a graph showing the production of anti-HIV-1 envelope binding antibodies upon immunization. Envelope-specific titers of bound antibodies in animals J408 and H445 were determined for the vaccine, ie, purified SF162ΔV2 gp140 oligomer protein, throughout the immunization scheme. The dotted line indicates the time of immunization, and the arrow indicates the time of virus attack.
FIG. 2
It is a graph which shows production of an HIV-1 neutralizing antibody. The presence of neutralizing antibodies against the homologous SF162ΔV2 virus and the parental SF162 virus was determined at various times during the immunization program. ：: before bleeding; ：: one month after the third DNA immunization; ■: two weeks after the first 'boost'; and ◆: two weeks after the second 'boost'.
FIG. 3
CD8 + T lymphocyte depletion: CD8 + T lymphocytes are depleted from vaccinated animals by bolus injection of anti-CD8 MAb OKT8F (arrow). Circulating CD4 + (dark circles), CD8 + T (white circles) and total CD3 + T from vaccinated and non-vaccinated animals in samples collected at various time points before and after SHIV 162P4 challenge (dotted line). The number of lymphocytes (stars) was determined.
FIG. 4A-B
Figure 4 is a graph showing viral load and production of anti-HIV envelope antibody titers after exposure to SHIV162P4. (A) Virus load is shown as number of RNA copies per ml of plasma. The dashed line indicates the limit of detection for this analysis (<500 copies / ml). Cross: unvaccinated animal AT54 was euthanized 111 days after the onset of SAAIDS after challenge. Arrows indicate when CD8 + cells reappear in the periphery of the vaccinated animal. (B) Production of anti-HIV envelope antibodies after SH162P4 challenge was monitored by an ELISA based on SF162ΔV2 gp140. Endpoint ELISA titers are shown.
FIG. 5
FIG. 2 shows the seroconversion of animals to SIV-gag / pol antigen and HIV env antigen in vaccinated and unvaccinated monkeys.
FIG. 6
FIG. 4 is a diagram showing antibody production in rabbits. The production of the anti-envelope antibody was determined by ELISA. Six animals (A1-A6) were immunized with DNA expressing the unmodified SF162 gp140 immunogen and six (A7-A12) with DNA expressing the modified ΔV2gp140 immunogen. Two weeks after each immunization, titers were determined by ELISA using oligomeric proteins of SF162 gp140 and ΔV2gp140. Dotted lines indicate the time points of each immunization.
FIG. 7a
It is a graph which shows neutralization of SF162 (DELTA) V2 virus and SF162 virus by rabbit serum. Shown are results from neutralization experiments using sera collected after the third and fifth immunizations against SF162ΔV2 (A) and SF162 (B) viruses. Data are representative of at least three independent experiments. The symbols indicate the mean% neutralization and standard deviation from three experimental wells. Dotted lines indicate 50%, 70% and 90% inhibition of infection. Dotted lines and asterisks (controls) are neutralization curves obtained with sera collected from animals immunized with DNA vector only, indicating non-specific neutralization.
FIG. 7B
It is a graph which shows neutralization of SF162 (DELTA) V2 virus and SF162 virus by rabbit serum. Shown are results from neutralization experiments using sera collected after the third and fifth immunizations against SF162ΔV2 (A) and SF162 (B) viruses. Data are representative of at least three independent experiments. The symbols indicate the mean% neutralization and standard deviation from three experimental wells. Dotted lines indicate 50%, 70% and 90% inhibition of infection. Dotted lines and asterisks (controls) are neutralization curves obtained with sera collected from animals immunized with DNA vector only, indicating non-specific neutralization.
FIG. 8
It is a graph which shows antibody production in a rhesus monkey. In animals immunized with the modified ΔV2gp140 immunogen (J408 and H445) and two animals immunized with the unmodified SF162gp140 immunogen (P655 and N472), and in control animals immunized with the DNA vector alone (M844 and H473) The production of anti-envelope antibodies was determined by ELISA using the corresponding proteins. The dotted line indicates the time of immunization. DNA: Animals were immunized three times monthly with a DNA vector expressing the gp140 form of each immunogen. Control animals received only the DNA vector. DNA + protein: The animals were immunized four times and simultaneously immunized with the corresponding CHO-producing gp140 oligomer protein using MF-59C adjuvant. Control animals received only adjuvant.
FIG. 9A
It is a graph which shows the neutralization activity of rhesus monkey serum. Serum collected from animals immunized with the modified ΔV2gp140 (A) and unmodified (B) SF162gp140 immunogens was determined for neutralizing activity against SF162 and SF162ΔV2 viruses as described in Example 2. Dotted lines indicate 50%, 70% and 90% inhibition of infection. Results are representative of 3-5 independent experiments. The data show the average and standard deviation from wells of three experiments. Before bleeding (Pre-bleed): serum collected before the start of vaccination; second and third DNA: serum collected one month after the second and third DNA administration, respectively; 2 weeks and 4 weeks after stimulation: sera collected 2 weeks and 4 weeks after DNA + protein 'booster' immunization, respectively.
FIG. 9B
It is a graph which shows the neutralization activity of rhesus monkey serum. Serum collected from animals immunized with the modified ΔV2gp140 (A) and unmodified (B) SF162gp140 immunogens was determined for neutralizing activity against SF162 and SF162ΔV2 viruses as described in Example 2. Dotted lines indicate 50%, 70% and 90% inhibition of infection. Results are representative of 3-5 independent experiments. The data show the average and standard deviation from wells of three experiments. Before bleeding (Pre-bleed): serum collected before the start of vaccination; second and third DNA: serum collected one month after the second and third DNA administration, respectively; 2 weeks and 4 weeks after stimulation: sera collected 2 weeks and 4 weeks after DNA + protein 'booster' immunization, respectively.
FIG. 10
Figure 4 is a graph showing neutralization of heterologous Clade B HIV-1 primary isolate by monkey serum. Neutralizing activity of the sera collected at 2 weeks and 4 weeks after DNA + protein 'booster' immunization against the vaccine heterologous HIV-1 primary isolate was determined as described in Example 2. Dotted lines indicate 50%, 70% and 90% inhibition of infection. The numbers indicate specific neutralization and are defined as the difference between the% virus neutralization recorded with sera collected after vaccination and the% virus neutralization recorded with sera collected before the start of vaccination. Data points represent the average% specific neutralization from two independent experiments.
FIG. 11A
FIG. 4 is a graph showing the production of bound and neutralizing antibodies after a second 'boost' immunization with a modified ΔV2gp140 protein. (A) The production of anti-envelope antibodies in two rhesus monkeys (J408 and H445) vaccinated with the modified ΔV2gp140 was determined by ELISA as described in Example 2. The dotted line indicates the time of immunization. DNA: The immunogen was immunized three times monthly with a DNA vector expressing the gp140 form. DNA + protein: Animals received a fourth round of DNA immunization and purified ΔV2gp140 oligomeric protein. Protein: Animals were immunized with purified ΔV2gp140 oligomer protein only. (B) Neutralizing activity of sera after the second 'boost' on SF162ΔV2 isolates and SF162 isolates was compared to sera collected after the first 'boost' (see Figure 4). Non-specific neutralization recorded in the pre-immune serum (pre-bleed) is also shown.
FIG. 11B
FIG. 4 is a graph showing the production of bound and neutralizing antibodies after a second 'boost' immunization with a modified ΔV2gp140 protein. (A) The production of anti-envelope antibodies in two rhesus monkeys (J408 and H445) vaccinated with the modified ΔV2gp140 was determined by ELISA as described in Example 2. The dotted line indicates the time of immunization. DNA: The immunogen was immunized three times monthly with a DNA vector expressing the gp140 form. DNA + protein: Animals received a fourth round of DNA immunization and purified ΔV2gp140 oligomeric protein. Protein: Animals were immunized with purified ΔV2gp140 oligomer protein only. (B) Neutralizing activity of sera after the second 'boost' on SF162ΔV2 isolates and SF162 isolates was compared to sera collected after the first 'boost' (see Figure 4). Non-specific neutralization recorded in the pre-immune serum (pre-bleed) is also shown.
FIG. 12A-B
FIG. 4 is a graph showing the presence of anti-V3 loop antibodies in serum collected from monkeys (macaques) immunized with a modified ΔV2gp140 immunogen. The production of anti-V3 loop antibodies was determined by using an ELISA method with V3 loop peptides from the SF162 / SF162ΔV2 envelope. (A) First, it was examined whether the captured V3 loop peptide interacts with a specific anti-V3 loop MAb that recognizes a linear (447D) V3 epitope and a conformational (391-95D) V3 loop epitope. (B) Next, the titers of anti-V3 loop antibodies present in the serum collected from the two vaccinated animals 2 and 4 weeks after the first and second boosters were determined. As a comparison, the titers of all anti-envelope antibodies present in the same serum were also included.
FIG. 13A
FIG. 4 is a graph showing neutralization of HIV-1 of clades A, E and D by sera from two animals immunized with a modified envelope protein from an HIV-1 clade B immunogen with a V2 region deletion.
FIG. 13B
FIG. 4 is a graph showing neutralization of HIV-1 of clades A, E and D by sera from two animals immunized with a modified envelope protein from an HIV-1 clade B immunogen with a V2 region deletion.
FIG. 14
This is an amino acid sequence showing the polynucleotide sequence of full-length SF162ΔV2 gp140 envelope protein (SEQ ID NO: 1).
FIG.
This is an amino acid sequence showing the polynucleotide sequence of SF162ΔV2 gp140 envelope protein fragment (SEQ ID NO: 3).
FIG.
This is an amino acid sequence showing the full-length SF162ΔV2 gp140 envelope protein (SEQ ID NO: 2).
FIG.
This is an amino acid sequence showing an SF162ΔV2 gp140 envelope protein fragment (SEQ ID NO: 4).

Claims (27)

A method of immunizing an animal against heterologous HIV-1, comprising at least one modified HIV-1 envelope protein or fragment thereof, or at least one modified HIV-1 envelope protein, wherein the V2 region is deleted. Administering to said animal an immunogen comprising a DNA or virus encoding the fragment, or a combination thereof, wherein said animal is immunized against at least one strain of HIV-1 other than said immunogen. The method. 2. The method of claim 1, wherein said immunity comprises a humoral response. 3. The method of claim 1 or 2, wherein the immunogen comprises a modified HIV-1 envelope protein from Clade B strain HIV-1. The method according to any one of claims 1 to 3, wherein the HIV-1 strain is SF162. The method according to any one of claims 1 to 4, wherein the modified HIV-1 envelope protein is SEQ ID NO: 2 or SEQ ID NO: 4. 5. The method of any one of claims 1-4, wherein the DNA encoding at least one of the modified HIV-1 envelope proteins is SEQ ID NO: 1 or SEQ ID NO: 3. 7. The method of any one of claims 2 to 6, wherein the humoral response comprises a neutralizing antibody. 8. The method of any one of claims 2 to 7, wherein the humoral response comprises a protective antibody. The method according to any one of claims 1 to 8, wherein the animal is a human. A method for eliciting a heterologous immune response to HIV-1 in an animal, comprising at least one modified HIV-1 envelope protein or fragment thereof, or at least one modified HIV-1 envelope protein, having a V2 region deletion. Or immunizing said animal with an immunogen comprising a DNA or virus encoding a fragment thereof, or a combination thereof, wherein said animal is enveloped by at least one strain of HIV-1 other than said immunogen. Such a method, which exhibits a specific immune response. 11. The method of claim 10, wherein said envelope-specific immune response comprises a humoral response. 12. The method of claim 10 or 11, wherein the immunogen comprises a modified HIV-1 envelope protein from Clade B strain HIV-1. The method according to any one of claims 10 to 12, wherein the HIV-1 strain is SF162. 14. The method according to any one of claims 10 to 13, wherein the modified HIV-1 envelope protein is SEQ ID NO: 2 or SEQ ID NO: 4. 14. The method according to any one of claims 10 to 13, wherein the DNA encoding at least one of the modified HIV-1 envelope proteins is SEQ ID NO: 1 or SEQ ID NO: 3. 16. The method of any one of claims 11 to 15, wherein the humoral response comprises a neutralizing antibody. 17. The method according to any one of claims 11 to 16, wherein the humoral response comprises a protective antibody. 18. The method according to any one of claims 10 to 17, wherein the animal is a human. A pharmaceutical composition for immunizing an animal against the HIV-1 virus, comprising at least one modified HIV-1 envelope protein or fragment thereof, or at least one modified HIV-I, wherein there is a V2 region deletion. (1) The pharmaceutical composition, comprising a DNA or virus encoding an envelope protein or a fragment thereof, or a combination thereof, which is effective for inducing a heterologous envelope-specific immune response, and a pharmaceutically acceptable carrier or excipient. 20. The pharmaceutical composition according to claim 19, wherein said modified HIV-1 envelope protein is derived from Clade B strain HIV-1. The pharmaceutical composition according to claim 19 or 20, wherein the HIV-1 strain is SF162. 22. The pharmaceutical composition according to claim 19, wherein the modified HIV-1 envelope protein is SEQ ID NO: 2 or SEQ ID NO: 4. 22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the DNA encoding at least one of the modified HIV-1 envelope proteins is SEQ ID NO: 1 or SEQ ID NO: 3. A method for evaluating whether a compound is capable of producing a protective antibody against at least one heterologous HIV-1 strain in an animal capable of producing a protective antibody against wild-type HIV-1, comprising immunizing said animal with said compound. The method, comprising the steps of: depleting the animal for CD8 + T lymphocytes; and assessing the animal for the presence of protective antibodies against at least one heterologous HIV-1 strain. 25. The method of claim 24, wherein said depleting is performed by administering an anti-CD8 monoclonal antibody to said animal. The method according to claim 24 or 25, wherein the compound is an HIV-derived polypeptide or a fragment thereof, or a DNA or virus encoding the polypeptide or a fragment thereof. 27. The method of any one of claims 24-26, wherein said immunizing is performed with a DNA vaccine, a protein, or a combination thereof.

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